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Introduction An increase intolerance to weight-bearing or ambulation was noted when second degree burns were sustained in the lower extremities, specifically knee distally. Patients were noted to request additional pain medication and/or additional time in the bed, which are not this burn center’s burn rehabilitation activity protocol. Use of double ace wraps, from knee distally, was initiated to increase patient’s participation in burn rehabilitation therapy. A retrospective chart review was used to determine if there were similar factors could be identified to anticipate use of a lower extremity double ace wrap. Methods Retrospective chart review from July 2020 to August 2021 was performed to identify lower extremity burns. Inclusion criteria included: age 13 or greater; admitted to this burn center w/LOS greater than 24 hours, received burn rehabilitation therapy; second and third degree burns located below the knee; and pre-admission status of independent ambulator. Chart review included 104 patients, with criteria met by 19 males and 5 females. The average age was 38.9 years with a range of 13 to 78 years. Length of stay ranged from 1 to 30 days and pre-exiting medical history included diabetes, type II in 8.3% and hypertension in 16.7% of the patients. The TBSA ranged from 1.15% to 42%. Surgical procedures of BWE w/dermal/epidermal skin substitute application from knee distally, occurred in 50% of the patients; 37.5% underwent BWE w/allograft and within 5-7 days underwent meshed split thickness skin grafts; 4% underwent BWE with placement of xeroform. 8.3% did not require surgery. Results All patients involved in the study required use of double ace wraps during hospital stay. Duration of use of double ace wraps depended on subjective report of abnormal sensations. Use was required mainly in the acute phase of burn injury prior to autografting, in partial thickness burns that underwent surgical procedure for application of dermal/epidermal skin substitute, or in patients that did not require autografting. Patient’s verbalizations of abnormal sensations with attempts to immediately weight bear or ambulate included: shooting pain, stinging pain, throbbing sensation, pins and needles. Reports of abnormal sensations and increased compliance with burn rehabilitation therapy was noted immediately following application of double ace wrap. Conclusions Burn Rehabilitation therapy protocol now includes close identification of superficial or deep second degrees located distally to the knee and patient’s verbal descriptions of abnormal sensations with attempts to place feet into a dependent position. Burn Rehabilitation therapy has developed a wearing schedule of the double ace wrap which has resulted in no adverse effects and positive patient compliance and participation.

Under summer-like photoperiods (15∶9 L∶D) with abundant high-quality food, larval growth and development of the baetid mayflies (Ephemeroptera) Cloeon dipterum-IS1 and C. dipterum-CT1 were ...

To better understand the effects of transient thermal stress in an aquatic insect, we first identified static temperatures associated with fitness deficits, and then reared larvae from egg hatch to adulthood under diurnally variable regimens including daily forays into deleterious temperatures. We sampled mature larvae at the coolest and warmest portions of their respective regimens for RNA-seq analysis. Few transcripts (28) were differentially expressed when larvae oscillated between favorable temperatures, while 614 transcripts were differentially expressed when experiencing daily transient thermal stress. Transcripts associated with N-glycan processing were downregulated while those associated with lipid catabolism and chitin turnover were significantly upregulated in heat stressed larvae. An across-regimen comparison of differentially expressed transcripts among organisms sampled at comparable temperatures demonstrated that the effects of daily thermal stress persisted even when larvae were sampled at a more optimal temperature (806 differentially expressed transcripts). The chronically stressed population had reduced expression of transcripts related to ATP synthesis, mitochondrial electron chain functions, gluconeogenesis and glycolytic processes while transcripts associated with cell adhesion, synaptic vesicle transport, regulation of membrane potential and lipid biosynthesis increased. Comparisons of constant vs. variable temperatures revealed that the negative consequences of time spent at stressful temperatures were not offset by more time spent at optimal temperatures.

We reared Cloeon dipterum from egg hatch to adult at 10 constant temperatures (12.1–33.5°C) to test 3 hypotheses (thermal equilibrium hypothesis, temperature size rule [TSR], and O2- and ca...

Understanding species' thermal limits and their physiological determinants is critical in light of climate change and other human activities that warm freshwater ecosystems. Here, we ask whether oxygen limitation determines the chronic upper thermal limits in larvae of the mayfly Neocloeon triangulifer, an emerging model for ecological and physiological studies. Our experiments are based on a robust understanding of the upper acute (∼40°C) and chronic thermal limits of this species (>28°C, ≤30°C) derived from full life cycle rearing experiments across temperatures. We tested two related predictions derived from the hypothesis that oxygen limitation sets the chronic upper thermal limits: (1) aerobic scope declines in mayfly larvae as they approach and exceed temperatures that are chronically lethal to larvae; and (2) genes indicative of hypoxia challenge are also responsive in larvae exposed to ecologically relevant thermal limits. Neither prediction held true. We estimated aerobic scope by subtracting measurements of standard oxygen consumption rates from measurements of maximum oxygen consumption rates, the latter of which was obtained by treating with the metabolic uncoupling agent carbonyl cyanide-4-(trifluoromethoxy) pheylhydrazone (FCCP). Aerobic scope was similar in larvae held below and above chronic thermal limits. Genes indicative of oxygen limitation (LDH, EGL-9) were only upregulated under hypoxia or during exposure to temperatures beyond the chronic (and more ecologically relevant) thermal limits of this species (LDH). Our results suggest that the chronic thermal limits of this species are likely not driven by oxygen limitation, but rather are determined by other factors, e.g. bioenergetics costs. We caution against the use of short-term thermal ramping approaches to estimate critical thermal limits (CTmax) in aquatic insects because those temperatures are typically higher than those that occur in nature.

Arsenic is an important environmental pollutant whose speciation and mobility in freshwater food webs is complex. Few studies have characterized uptake and efflux rates of arsenic in aquatic benthic invertebrates. Further, we lack a fundamental understanding of how pH influences uptake kinetics in these organisms or how this key environmental variable could alter dietary exposure for primary consumers. Here we used a radiotracer approach to characterize arsenate accumulation dynamics in benthic invertebrates, the influence of pH on uptake in a subset of these organisms, and the influence of pH on uptake of arsenate by periphyton - an important food source at the base of aquatic food webs. Uptake rate constants (K u ) from aqueous exposure were modest, ranging from ∼0.001 L g −1 d −1 in three species of mayfly to 0.06 L g −1 d −1 in Psephenus herricki . Efflux rate constants ranged from ∼0.03 d −1 in Corbicula fluminea to ∼0.3 d −1 in the mayfly Isonychia sp , and were generally high. Arsenate uptake decreased with increasing pH, which may be a function of increased adsorption at lower pHs. A similar but much stronger correlation was observed for periphyton where K u decreased from ∼3.0 L g −1 d −1 at 6.5 pH to ∼0.7 L g −1 d −1 at 8.5 pH, suggesting that site specific pH could significantly alter arsenic exposure, particularly for primary consumers. Together, these findings shed light on the complexity of arsenic bioavailability and help explain observed differences reported in the literature.

The study of processes driving diversification requires a fully sampled and well resolved phylogeny, although a lack of phylogenetic markers remains a limitation for many non-model groups. Multilocus approaches to the study of recent diversification provide a powerful means to study the evolutionary process, but their application remains restricted because multiple unlinked loci with suitable variation for phylogenetic or coalescent analysis are not available for most non-model taxa. Here we identify novel, putative single-copy nuclear DNA (nDNA) phylogenetic markers to study the colonization and diversification of an aquatic insect species complex, Cloeon dipterum L. 1761 (Ephemeroptera: Baetidae), in Macaronesia. Whole-genome sequencing data from one member of the species complex were used to identify 59 nDNA loci (32,213 base pairs), followed by Sanger sequencing of 29 individuals sampled from 13 islands of three Macaronesian archipelagos. Multispecies coalescent analyses established six putative species. Three island species formed a monophyletic clade, with one species occurring on the Azores, Europe and North America. Ancestral state reconstruction indicated at least two colonization events from the mainland (to the Canaries, respectively Azores) and one within the archipelago (between Madeira and the Canaries). Random subsets of the 59 loci showed a positive linear relationship between number of loci and node support. In contrast, node support in the multispecies coalescent tree was negatively correlated with mean number of phylogenetically informative sites per locus, suggesting a complex relationship between tree resolution and marker variability. Our approach highlights the value of combining genomics, coalescent-based phylogeography, species delimitation, and phylogenetic reconstruction to resolve recent diversification events in an archipelago species complex.

An inability to adequately meet tissue oxygen demands has been proposed as an important factor setting upper thermal limits in ectothermic invertebrates (especially aquatic species) as well as explaining the observed decline in adult size with increased rearing temperature during the immature stages (a phenomenon known as the Temperature Size Rule, or TSR). We tested this by rearing three aquatic insects (the mayflies Neocloeon triangulifer and two species of the Cloeon dipterum complex) through their entire larval life under a range of temperature and oxygen concentrations. Hyperoxia did not extend upper thermal limits, nor did it prevent the loss of size or fertility experienced near upper chronic thermal limits. At moderate temperatures, the TSR pattern was observed under conditions of hyperoxia, normoxia, and hypoxia, suggesting little or no influence of oxygen on this trend. However, for a given rearing temperature, adults were smaller and less fecund under hypoxia due to a lowering of growth rates. These mayflies greatly increased the size of their gills in response to lower dissolved oxygen concentrations but not under oxygen-saturated conditions over a temperature range yielding the classic TSR response. Using ommatidium diameter as a proxy for cell size we found the classic TSR pattern observed under moderate temperature conditions was due primarily to a change in the number of cells rather than cell size. We conclude overall that a failure to meet tissue oxygen demands is not a viable hypothesis for explaining either the chronic thermal limit or TSR pattern in these species.

Phyllopalpus pulchellus Uhler (Trigonidiinae) is a small, diurnally-active cricket common throughout much of the eastern United States. Mating interactions in this species are here described based on observations of 14 mating pairs. Males produce two very different types of spermatophores: small, spermless microspermatophores which are removed and consumed by females shortly after transfer, and larger, sperm-containing macrospermatophores that are only produced following the successful transfer of a microspermatophore. A bell-shaped structure surrounding the sperm tube of macrospermatophores makes them more difficult for females to remove prematurely. Males' production and transfer of spermless microspermatophores likely serve as a test of female receptivity prior to investing in a relatively expensive macrospermatophore that must be used within a short period.

We assessed water quality using physical, chemical, and biological characteristics in 37 streams between Puerto Maldonado and Cusco in Peru. Study sites ranged from ∼200 to 4000 m in elevation, with streams selected as pairs (control/natural vs. human impacted) along this gradient. In general, temperature decreased (R2 = 0.82) and other parameters increased [dissolved oxygen (R2 = 0.19), conductivity (R2 = 0.17), pH (R2 = 0.37)] with elevation. Macroinvertebrates were hand collected by professional entomologists and using leaf packs implemented by conservation workers. The professionals identified 213 unique taxa from the hand collections, with 80 species collected only one time. Data from control streams showed that as elevation increased, total richness (p = 0.008) and EPT richness (p = 0.050) decreased whereas Diptera richness increased (p = 0.002). NMS ordination indicated significant differences in macroinvertebrate assemblages when control streams from low and high elevations were contrasted. Hand collections also revealed significant differences between control and impact streams for total richness, EPT richness and % Diptera, but not for % EPT, % EPT richness, or % Diptera richness. The majority of the deployed leaf packs were successfully retrieved and contained many macroinvertebrates (Avg. = 141 individuals per pack). There were 98 unique taxa (family level or higher) identified from the leaf packs by the trained conservation workers. Fourteen of 15 macroinvertebrate community metrics (at the family level) were able to detect significant differences between control and impact sites. All of the family level metrics responded similarly across the elevation gradient except total richness, EPT richness, EPT count, and % Hydropsychids. Both the Costa Rica and the Virginia Save Our Stream Indexes were able to differentiate control from impacted streams using leaf pack data. Although the diversity of macroinvertebrates was higher for hand collections relative to leaf packs (due to greater habitats sampled and higher taxonomic resolution), leaf pack samples were better able to distinguish control from impacted streams. Specifically, leaf packs were able to discern impacts in streams at low elevation better than those at high elevation. Generally, macroinvertebrates indicated impact from urbanization to be worse than impacts from other human activities (i.e., gold mining and agriculture). Overall, hand collections will serve as an important reference of species diversity going forward while leaf packs processed by trained conservation workers are a viable method to monitor stream water quality in Peru and perhaps elsewhere.

Salinity in freshwater ecosystems has increased significantly at numerous locations throughout the world, and this increase often reflects the use or production of salts from road de-icing, mining/oil and gas drilling activities, or agricultural production. When related to de-icing salts, highest salinity often occurs in winter when water temperature is often low relative to mean annual temperature at a site. Our study examined acute (96 h) responses to elevated salinity (NaCl) concentrations at five to seven temperature treatments (5–25°C) for four mayfly species (Baetidae: Neocloeon triangulifer , Procloeon fragile ; Heptageniidae: Maccaffertium modestum ; Leptophlebiidae: Leptophlebia cupida ) that are widely distributed across eastern North America. Based on acute LC50s at 20°C, P. fragile was most sensitive (LC50 = 767 mg l −1 , 1447 µS cm −1 ), followed by N. triangulifer (2755 mg l −1 , 5104 µS cm −1 ), M. modestum (2760 mg l −1 , 5118 µS cm −1 ) and L. cupida (4588 mg l −1 , 8485 µS cm −1 ). Acute LC50s decreased as temperature increased for all four species ( n = 5–7, R 2 = 0.65–0.88, p = 0.052–0.002). Thus, acute salt toxicity is strongly temperature dependent for the mayfly species we tested, which suggests that brief periods of elevated salinity during cold seasons or in colder locations may be ecologically less toxic than predicted by standard 20 or 25°C laboratory bioassays. This article is part of the theme issue ‘Salt in freshwaters: causes, ecological consequences and future prospects’.

Criteria for establishing water quality standards that are protective for 95% of the native species are generally based upon laboratory toxicity tests. These tests utilize common model organisms that have established test methods. However, for invertebrates these species represent mostly the zooplankton community and are not inclusive of all taxa. In order to examine a potential under-representation in emerging aquatic invertebrates the US Environmental Protection Agency has cultured a parthenogenetic mayfly, Centroptilum triangulifer (Ephemeroptera: Baetidae). This study established a 48h acute and a 14-day short-term chronic testing procedure for C. triangulifer and compared its sensitivity to two model invertebrates, Ceriodaphnia dubia and Daphnia magna. Toxicity tests were conducted to determine mortality and growth effects using standard reference toxicants: NaCl, KCl and CuSO4. In 48-h acute tests, the average LC50 for the mayfly was 659mgL(-1) NaCl, 1957mgL(-1) KCl, and 11μgL(-1) CuSO4. IC25 values, using dry weight as the endpoint, were 228mgL(-1) NaCl, 356mgL(-1) KCl and 5μgL(-1) CuSO4. C. triangulifer was the most sensitive species in NaCl acute and chronic growth tests. At KCl concentrations tested, C. triangulifer was less sensitive for acute tests but was equally or more sensitive than C. dubia and D. magna for growth measurements. This study determined C. triangulifer has great potential and benefits for use in ecotoxicological studies.

We develop a three-stage stochastic individual-based model for the phenology of the parthenogenetic mayfly Neocloeon triangulifer (Ephemeroptera: Baetidae). Mayflies are poikilothermic and develop at a rate dependent on the temperature of their aquatic environment. The model was built using development data for each aquatic stage from controlled laboratory studies to determine the variation in development rates present in the population and the development rate response curve to temperature. Variable temperature experiments simulating diel temperature changes were used to validate the model. The thermal input data for the model is based on average daily water temperature recordings from White Clay Creek from January 2007 to 2013. The model predicts a multivoltine life cycle with a mode of 3 generations per year that agrees well with field observations. An optimally timed quiescent period for larvae (triggered by day length) is shown to enhance synchronization of adult emergence. Any future stream temperature rise is predicted to increase the number of generations per year and desynchronize adult emergence, and these changes could lead to significant population decline in species that reproduce sexually.

Anthropogenic disturbances, including those from developing energy resources, can alter stream chemistry significantly by elevating total dissolved solids. Field studies have indicated that mayflies (Order Ephemeroptera) are particularly sensitive to high total dissolved solids. In the present study, the authors measured 20-d growth and survivorship of larval Neocloeon triangulifer exposed to a gradient of brine salt (mixed NaCl and CaCl2) concentrations. Daily growth rates were reduced significantly in all salt concentrations above the control (363 µS cm−1) and larvae in treatments with specific conductance >812 µS cm−1 were in comparatively earlier developmental stages (instars) at the end of the experiment. Survivorship declined significantly when specific conductance was >1513 µS cm−1 and the calculated 20-d 50% lethal concentration was 2866 µS cm−1. The present study's results provide strong experimental evidence that elevated ion concentrations similar to those observed in developing energy resources, such as oil and gas drilling or coal mining, can adversely affect sensitive aquatic insect species. Environ Toxicol Chem 2015;34:167–172. © 2014 SETAC

Molting is a stressful event in insect development. When an insect molts, the individual discards its exoskeleton and sheds and renews the interior lining of substantial portions of the respiratory (tracheal) system. We profiled for the first time the disruptive pattern of respiration during the molting process in larvae of the mayfly Cloeon dipterum (Ephemeroptera:Baetidae). Molting induces a precipitous drop in O2 consumption immediately followed by a surge in O2 consumption that appears to be compensatory in nature. Postmolt metabolic suppression is consistently observed during which O2 consumption rates lag relative to those of nonmolting larvae. Furthermore, the magnitude of respiratory disturbance during the molt increases as a function of temperature. Increasing temperatures increase molting frequency and the apparently stressful nature of the molt itself. Thus, the insect molt appears to be a previously unappreciated route by which warming conditions may affect aquatic insects.

The genus Anaxipha has at least 13 North American species, eight of which are described here. Ten species fall into these three species groups: exigua group (exigua Say, scia Hebard and n. spp. thomasi, tinnulacita, tinnulenta, and tinnula); delicatula group (delicatula Scudder and vernalis n. sp.); litarena group (litarena Fulton and rosamacula n.sp.). The remaining three (imitator Saussure, fultoni n.sp., and calusa n.sp.) have no close relatives among the other species. Most new species were initially distinguished by their calling songs, and in most cases sympatric populations proved cleanly separable by features of male genitalia and tooth-counts of stridulatory files. Species groups were based mostly on comparisons of male genital structures and the results of DNA barcoding. Species are here characterized not only by their songs and morphology, but also by geographical, ecological, and seasonal distributions. At a given temperature the pulse rate (PR) of the male's calling song is a key aid...

Understanding species' thermal limits and their physiological determinants is critical in light of climate change and other human activities that warm freshwater ecosystems. Here, we ask whether oxygen limitation determines the chronic upper thermal limits in larvae of the mayfly

Periphyton is an important food source at the base of freshwater ecosystems that tends to bioconcentrate trace elements making them trophically available. The potential for arsenic-a trace element of particular concern due to its widespread occurrence, toxicity, and carcinogenicity-to bioconcentrate in periphyton and thus be available to benthic grazers is less well characterized. To better understand arsenate bioaccumulation dynamics in lotic food webs, we used a radiotracer approach to characterize accumulation in periphyton and subsequent trophic transfer to benthic grazers. Periphyton bioconcentrated As between 3,200-9,700-fold (dry weight) over 8 days without reaching steady state, suggesting that periphyton is a major sink for arsenate. However, As-enriched periphyton as a food source for the mayfly Neocloeon triangulifer resulted in negligible As accumulation in a full lifecycle exposure. Additional studies estimate dietary assimilation efficiency in several primary consumers ranging from 22% in the mayfly N. triangulifer to 75% in the mayfly Isonychia sp. X-ray fluorescence mapping revealed that As was predominantly associated with iron oxides in periphyton. We speculate that As adsorption to Fe in periphyton may play a role in reducing dietary bioavailability. Together, these results suggest that trophic movement of As in lotic food webs is relatively low, though species differences in bioaccumulation patterns are important.

Mayflies (Ephemeroptera) are important to the food web of most stream, river, and lake ecosystems and are critical to water-quality monitoring programs. They are widespread and ancient (predate dinosaurs) and have primitive reproductive systems and the shortest adult life spans of all insects. Here we formulate and test the hypothesis that facultative parthenogenesis occurs as a widespread adaptation in most, if not all, mayflies. A rare form of reproduction, facultative parthenogenesis combines the short-term advantages of parthenogenesis, which doubles reproductive output, with the long-term advantages of genetic variation associated with sex. For 7 species of bisexual mayflies, we show that their eggs hatched whether fertilized or not and that unfertilized eggs took longer to develop and hatch. However, once hatched, larvae produced via parthenogenesis grew and developed to the adult in a manner similar to that of larvae produced sexually. In addition, for all study species, female adults produ...

In 2006, the Stroud Water Research Center conducted inventories of stream macroinvertebrates in the Peninsula de Osa in Costa Rica and the Madre de Dios watershed in eastern Peru. Both areas have extensive lowland tropical rainforests under threat from road development, tourism, poaching and gold mining. The mayfly communities of the two regions were substantially different in family relative abundances. In Osa the mayfly community was more or less evenly divided among Baetidae, Leptohyphidae, and Leptophlebiidae. In streams where one group was clearly dominant, this was most often Leptohyphidae. By contrast, in the Madre de Dios watershed Leptophlebiidae was often 75% or more of the mayfly fauna while Leptohyphidae was 20% or less. In both Osa and Madre de Dios, EPT indices were calculated for impacted streams and relatively undisturbed streams. However, physical characteristics such as stream size and substrate diversity were often a better predictor of community composition than human activity.

We compared genetic lineages in the mayfly genus Ephemerella (Ephemeroptera:Ephemerellidae) identified from mitochondrial DNA (mtDNA) to current taxonomy in 9 morphological taxa, including 2 geographically widespread species, Ephemerella invaria ( = E. inconstans, E. rotunda, E. floripara) and Ephemerella dorothea ( = E. infrequens). Maximum likelihood and parsimony analyses of the mtDNA sequences placed E. inconstans and E. invaria in a well-supported clade; however, mean Kimura 2-parameter genetic distance between the lineages was high (5.2%) relative to distance within lineages (1.3%). The phylogenetic relationships of synonyms E. rotunda and E. floripara were not resolved, but estimates of mean genetic distance to E. invaria were high for both (8.5% and 11.6%, respectively). Populations of E. dorothea were grouped in 2 well-supported clades (12.9% mean divergence) that did not include the synonym E. infrequens (20.9% mean divergence, based on a single sample). A large genetic distance (18.6%) ...

We examined Hg(II) bioaccumulation and compartmentalization patterns in conjunction with antioxidant responses in four aquatic insect species: two caddisflies (Chimarra sp. and Hydropsyche betteni) and two mayflies (Maccaffertium modestum and Isonychia sp). Total antioxidant capabilities differed among unexposed larvae, with both caddisfly species exhibiting elevated antioxidant activities relative to the mayflies. We were able to account for these differences by examining the constitutive activities of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), and superoxide dismutase (SOD), in the four species. We also examined levels of reduced and oxidized glutathione and cysteine in the insects. Glutathione peroxidase and SOD were the most responsive to Hg exposure, with GPx catalytic activity increasing between 50 and 310%. Superoxide dismutase activity decreased between 35 and 50%. This SOD suppression was shown to be dose-dependent in both caddisflies, butthe strength of this suppression did not appear to be related to rates of uptake. Surprisingly, little Hg (10%) was found in the heat-stable cytosolic protein subcellular compartment in each of the four species, suggesting that Hg was not well detoxified. By combining bioaccumulation studies with other physiological measures, we can begin to better understand the consequences of trace metal pollutants in nature.

For many years the Drunella lata complex of eastern North America consisted of 4 recognized species: 3 described from the Northeast, Drunella lata (Morgan), Drunella cornuta (Morgan), and Drunella cornutella (McDunnough), and 1, Drunella longicornis (Traver), from the southern Appalachian Mountains region. Recently, all 4 were synonymized to a single species (D. lata [Morgan], by priority) because morphological variation was perceived to be continuous and historical characterizations of species were viewed as arbitrary. We used genetic, morphometric, and life-history data to reevaluate D. lata, D. cornuta, and D. cornutella in northeastern North America, where all 3 morphotypes coexist in certain catchments and often in the same reach of stream. Pairwise genetic comparisons of coexisting morphotypes revealed fixed allelic differences at 6 to 10 of 19 allozyme loci, showing reproductive isolation among D. cornuta, D. cornutella, and D. lata even within the same reach of stream. Morphometric studies...

Eurylophella oviruptis new species is described and illustrated from female larvae and reared subimagos and imagos collected from swamp streams in North Carolina, USA. Several key morphological characters in the larva place this new species in the Eurylophella temporalis group. Genetic comparisons with other eastern North American Eurylophella revealed a fixation of alleles unique to E. oviruptis at 4 of 19 allozyme loci. Eurylophella oviruptis appears to be obligately parthenogenetic because no males were observed in the field or laboratory and eggs taken from subimagos and imagos hatched parthenogenetically (mean hatch rate = 79%). Abdomens of 60% of subimagos reared in the laboratory burst along the ecdysial line of the first 3 tergites immediately after transformation to the subimago at the water surface. Abdominal bursting ruptured the oviducts, released most of the eggs into the water, and left the subimago trapped on the water surface with a large, inflated midgut protruding from the split...

Allozymes were used to examine population genetic structure and species boundaries in the clonal, obligately parthenogenetic Centroptilum triangulifer and its sexual sister species Centroptilum alamance from 3 sites in Pennsylvania and 1 site in North Carolina, USA. Genotype frequencies in field populations of C. triangulifer showed numerous departures from Hardy-Weinberg expectations (mostly heterozygote excesses) and significant linkage disequilibrium at most testable locus combinations, as expected for a clonal parthenogen. A total of 51 distinct clones of C. triangulifer was identified, some of which were found at multiple sites (separated by .700 km in one case). Each stream contained from 7 to 25 clones and clonal frequencies at a given site varied greatly over time. Allelic patterns in laboratory hybrids suggest that parthenogenesis in C. triangulifer is diploid and automictic, and that crossing over is suppressed. In contrast, C. alamance populations had only a few Hardy-Weinberg departures (all heterzygote deficiences) and little or no linkage disequilibrium. No published work provides characters to distinguish the 2 species, but our data support retention of specific status for C. triangulifer and C. alamance because: 1) consistent, fixed allelic differences were found over a broad geographic area (.700 km) with no evidence of introgression despite co-occurrence (sympatry) in the same stream, 2) laboratory hybridization experiments demonstrated the existence of postzygotic barriers to gene flow between the species, and 3) measures of both interspecific and intraspecific genetic similarity were comparable to those found between other mayfly species. Phylogenetic analysis of the genetic data suggests speciation preceded the transition to obligate parthenogenetic reproduction in C. triangulifer. Morphological examination of genetically identified specimens enabled us to describe subtle but consistent differences that can be used to distinguish the species. Thus, what initially appeared to be single populations with skewed sex ratios (7, 11, and 24% male) at 3 of our study sites was, in fact, 2 populations at each site—1 exclusively female (C. triangulifer) and the other a normal, sexual (1:1 sex ratio) population (C. alamance). A similar approach, combining detailed genetic and breeding experiments with close morphological study, could help resolve taxonomic problems in some of the numerous other bisexual/parthenogenetic mayfly taxa.

Cyrtoxipha gundlachi and Orocharis gryllodes are two common, well-studied Florida crickets whose scientific names are in doubt. We determine that C. gundlachi Saussure (1874) may retain its name and designate a neotype to stabilize that interpretation. We find that Cyrtoxipha orientalis Desutter-Grandcolas (2003) is a valid species rather than a junior synonym of Cyrtoxipha gundlachi sensu Otte & Perez-Gelabert (2009) and that the Jamaican species C. clarki Otte & Perez-Gelabert (2009) is a close relative of C. gundlachi. Finally, we determine that the Florida species formerly known as O. gryllodes (Pallas 1772) is now Antillicharis oriobates Otte & Perez-Gelabert (2009).

Anthropogenic disturbances, including those from developing energy resources, can alter stream chemistry significantly by elevating total dissolved solids. Field studies have indicated that mayflies (Order Ephemeroptera) are particularly sensitive to high total dissolved solids. In the present study, the authors measured 20-d growth and survivorship of larval Neocloeon triangulifer exposed to a gradient of brine salt (mixed NaCl and CaCl2 ) concentrations. Daily growth rates were reduced significantly in all salt concentrations above the control (363 µS cm(-1) ) and larvae in treatments with specific conductance812 µS cm(-1) were in comparatively earlier developmental stages (instars) at the end of the experiment. Survivorship declined significantly when specific conductance was1513 µS cm(-1) and the calculated 20-d 50% lethal concentration was 2866 µS cm(-1) . The present study's results provide strong experimental evidence that elevated ion concentrations similar to those observed in developing energy resources, such as oil and gas drilling or coal mining, can adversely affect sensitive aquatic insect species.

Development of methods for assessing exposure and effects of waterborne toxicants on stream invertebrate species is important to elucidate environmentally relevant information. Current protocols for freshwater invertebrate toxicity testing almost exclusively utilize cladocerans, amphipods or chironomids rather than the more typical aquatic insect taxa found in lotic systems. Centroptilum triangulifer is a parthenogenetic mayfly occurring in depositional habitats of streams and rivers of the Eastern U.S. and Canada. C. triangulifer is an ideal stream insect for toxicity testing under field and laboratory conditions because of its short life cycle, parthenogenetic mode of reproduction, and it represents a group considered sensitive to environmental stressors. In this study, a colony of C. triangulifer was reared using a defined diet of three diatoms, Mayamaea atomus var. permitis, Nitzschia cf. pusilla, and Achnanthidium minutissimum. Percent survival (⩾80%), fecundity measurements (⩾1000 eggs) and pre-egg laying weights were used as indicators of overall colony health and fitness in our laboratory water (Lab-line) and in Moderately Hard Reconstituted Water (MHRW). Lab-line reared C. triangulifer had average survival rate of 92.69% for eleven generations and 82.99% over thirteen generations. MHRW reared C. triangulifer had an average survival rate of 80.65% for four generations and three generations of fecundities greater than 1000 eggs per individual. Pre-egg laying weight and fecundity were highly correlated and a best-fit model equation was derived to estimate egg counts for future generations. Establishment of this culturing protocol provides a more ecologically relevant species for toxicity testing and aids in further stressor identification for stream bioassessments.

Life history characteristics of the stream mayfly Euthyplocia hecuba (Hagen) (Polymitarcyidae:Euthyplociinae) were studied over a 4-yr period in two tropical streams (Rio Tempisquito and Quebrada Marilin) draining primary evergreen forest in northern Costa Rica. Larvae burrow in the substratum of the stream, living under small to large cobbles that are firmly embedded in the stream bottom. Euthyplocia hecuba is sexually dimorphic and one of the largest mayflies of Central America, with maximum larval size of 149 mg (dry mass) for females and 35 mg for males. Mature female and male larvae are about 40% larger in Q. Marilin relative to R. Tempisquito. Subimagoes emerge about 1 h before dawn (∼0400 h); the molt to the imago (or true adult stage) occurs ∼20 min later. Adult emergence and reproduction occurs mainly between June and November. Females mate and then oviposit in riffle areas just before dawn, with the time period gradually changing from 0446 and 0507 h in June to 0508 and 0529 h in October. We fou...

Diet is often the predominant route of trace metal exposure in aquatic insects. In freshwater ecosystems, periphyton serves as a primary source of food to many aquatic insects and is a major sink for trace metals. We investigated the bioconcentration of the essential metal Zn by periphyton using (65)Zn as a radiotracer. At relatively low dissolved concentrations (2-20 μg L(-1)), non steady state Zn bioconcentration by periphyton averaged 6,099 ± 2,430-fold, with much of the variability determined by loading regime (number of renewals and duration of exposures). Labeled periphyton was used as a food source for dietary accumulation studies with the mayfly Centroptilum triangulifer. After 29 days, larvae concentrated Zn 19-, 16- and 17-fold relative to dietary Zn concentrations of 8.1, 43.2 and 82.3 μg g(-1) (dry weight), respectively. Adults from that same cohort only concentrated Zn 8-, 3- and 3- fold relative to those same dietary concentrations, revealing that mayflies lose significant Zn prior to reaching adulthood. Anecdotal evidence suggests that this loss occurs prior to emergence to the subimago, as negligible Zn was found in the subimago to imago exuvium. Across a range of adult tissue concentrations, maternal transfer consistently averaged 26.7 %. Uptake (k(u), 0.26 L g(-1 )d(-1)) and efflux rate constants (k(e), 0.001-0.007 d(-1)) were measured and assimilation efficiencies from dietary Zn concentrations of 4.9 and 59.7 μg Zn g(-1) were estimated to be 88 ± 4 % and 64 ± 15 %, respectively. Both life cycle and biodynamic modeling approaches point towards diet being the primary route of Zn bioaccumulation in this mayfly.

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3–24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.

Walker (in Weissman, Walker, & Gray 2009) described a new species of Gryllus, G. jamaicensis, a second species recorded from Jamaica to the widespread G. assimilis (Fabricius 1775). The two taxa are apparent sister species and are morphologically indistinguishable. They can be separated by three calling song characteristics: pulses per chirp, average pulse rate, and dominant frequency.

Selenium effects in nature are mediated by the relatively large bioconcentration of aqueous Se by primary producers and smaller, yet critical, dietary transfers to primary consumers. These basal processes are then propagated through food webs to higher trophic levels. Here we quantified the movement of dissolved Se (as selenite) to periphyton, and used the resultant periphyton as a food source for conducting full life-cycle dietary Se exposures to the mayfly Centroptilum triangulifer. Periphyton bioconcentrated Se ~2,200-fold from solution in a log-linear fashion over dissolved Se concentrations ranging from 1.1 to 23.1 μg L(-1). We examined the influence of two feeding ration levels (1x and 2x) on trophic transfer, tissue Se concentrations, maternal transfer, and functional endpoints of mayfly performance. Mayflies fed a lesser ration (1x) displayed greater trophic transfer factors (mean TTF, 2.8 ± 0.4) than mayflies fed 2x rations (mean TTF, 1.1 ± 0.3). In 1x exposures, mayflies exhibited significant (p0.05) reductions in survivorship and total body mass at dietary [Se] ≥ 11.9 μg g(-1), reduced total fecundity at ≥ 4.2 μg g(-1), and delayed development at ≥ 27.2 μg g(-1). Mayflies fed a greater ration (2x) displayed reduced tissue Se concentrations (apparently via growth dilution) relative to 1x mayflies, with no significant effects on performance. These results suggest that the influence of Se on mayfly performance in nature may be tied to food resource availability and quality. Furthermore, nutritional status is an important consideration when applying laboratory derived estimates of toxicity to risk assessments for wild populations.

The stream mayfly Cloeon triangulifer is well suited as a bioassay organism because it has a relatively short egg and larval stage and can be readily cultured under laboratory conditions. Moreover, the species is widely distributed and reproduces as parthenogenetic clones. Nineteen distinct clones have been isolated from natural populations with Nei's genetic distance between clones ranging from 0.004 to 0.183. Clonal reproduction gives research scientists the option of including or excluding genetic variation from the experimental design. The present study involved exposing individuals from a single clone to initial technical chlordane levels ranging between 4.3 and 56.3 μg/L. No effect of chlordane was observed for the egg stage; eggs required 9 d to hatch at 20°C, and >95% of the eggs hatched in all controls and treatments. Egg hatch success was also tested at higher concentrations (89.0 and 158.3 μg/L), with no effect being observed. In contrast, larval survivorship decreased significantly from about 80% for control groups to 18.0 and 15.4 μg/L. Sublethal effects on larvae exposed to 4.3 μg/L of chlordane included significantly longer developmental time (37 vs. 34.8 d) and larger adult size (1.5 vs. 1.1 mg) relative to controls. Body burdens of chlordane in adult tissue for individuals reared in the 4.3-μg/L treatment ranged from 51 to 140 ppb. Chlordane-related compounds found in adult mayfly tissue were cis-nonachlor (6%), trans-nonachlor (45%), cis-chlordane (4%), trans-chlordane (5%), oxychlordane (21%), and heptachlor epoxide (18%).

Selenium contamination in aquatic ecosystems provides management challenges because bioaccumulation in animals is largely a function of dietary exposure, whereas regulatory entities have traditionally focused on direct water to organism interactions. Selenium is known to be readily absorbed by primary producers and can potentially biomagnify in food webs and elicit adverse effects in higher trophic levels. However, selenium bioaccumulation in the invertebrate prey of many predatory animals is poorly understood. Here, we used 75Se (as selenite) as a radiotracer to characterize Se bioaccumulation into natural periphyton biofilms and subsequent dietary and maternal transfer in the mayfly, Centroptilum triangulifer, in a life-cycle assay. On average periphyton biofilms bioconcentrated selenium 1113 (+/-430)-fold following 7-9 days of exposure to a range of environmentally relevant dissolved concentrations (2.4-13.9 microg L(-1)). Mayflies grown to adulthood on these diets further biomagnified Se with trophic transfer factors averaging 22 (+/-0.4)-fold in postpartum maternal tissues. Adults then transferred 46.5 (+/-8.8)% of their body burdens to eggs with an observed reduction in fecundity for mayflies fed on diets greater than approximately 11 microg g(-1). These results suggest that at environmentally feasible dietary Se concentrations insects are potentially affected by Se exposure, and that the current presumption that insects are simply conduits of Se to higher trophic levels is inaccurate.

In streams, periphyton biofilms are important sinks for trace metals such as cadmium and are primary food sources of many invertebrate consumers. To study Cd trophic transfer, we produced differentially contaminated diets by exposing natural periphyton to environmentally relevant dissolved Cd ranging from 0 to 10 μg L−1 for 6–7 days using a radiotracer approach. On average, periphyton grown during three different seasons bioconcentrated Cd similarly – approximately 1315 (±442) -fold above dissolved concentrations. However, mayfly larvae (Centroptilum triangulifer) raised on these differentially contaminated diets (first instar through adulthood) had significantly higher trophic transfer factors from periphyton grown in Aug and Nov 2008 (4.30 ± 1.55) than from periphyton grown in Jan 2009 (0.85 ± 0.21). This Cd bioaccumulation difference is only partially explained by apparent food quality and subsequent growth differences. Taken together, these results suggest that primary producers at the base of food webs drive metal bioaccumulation by invertebrate grazers.

The genetic structure of Dolania populations in South Carolina, Alabama, and Florida was examined using starch gel electrophoresis. All study populations except Alabama consisted entirely of D. americana Edmunds and Traver. Electrophoresis revealed that the Alabama site contained both D. americana and an undescribed species (D. sp. nov.), with the latter taxon predominating. Individuals of the two taxa had nearly fixed allelic differences at two loci (Est 4 and Gda). The extent of genetic differentiation between populations of the two taxa in the same river or between adjacent river systems was significantly greater than between D. americana populations separated by large geographic distances. Populations of both species were characterized by relatively normal levels of polymorphic loci (average = 23%) but unusually low levels of heterozygosity (average = 0.03). Genetic variation between the Florida and South Carolina populations of D. americana was statistically significant. There was also some indicatio...

We examined the function of secondary sexual characters in the role-reversed, lekking behaviour of female long-tailed dance flies, Rhamphomyia longicauda Loew (Empididae), to test the hypothesis that the degree of abdominal distention is an honest female signal about the state of egg development. Female Rhamphomyia cannot hunt for prey and they receive all of their protein from males by exchanging copulations for nuptial prey gifts. Females compete for male gifts within leks that are organized for a brief period each evening before dark. Before hovering within leks, females swallow air, inflating expandable pouches on the pleural margins of the abdomen. The result is a large saucer-like abdomen which is further exaggerated by wrapping scaled pro-, meso- and metathoracic legs along its pleural margins. Male preference for an enlarged abdomen was confirmed by suspending plastic models of varying size from monofilament lines and recording which models attracted the most males. There was a positive relationship between egg development and abdominal distention in a related species, R. sociabilis (Williston), which lacks inflatable abdominal pouches. Multiple regression showed that in R. longicauda, abdominal inflation completely masks the state of egg development. We conclude that female R. longicauda deceive mate-seeking males with the unreliable message that eggs are nearing maturation in order to obtain a protein meal in exchange for copulation. Males that fail to identify a female bearing mature eggs risk near-certain cuckoldry and an increased probability that the female will die before oviposition. Copyright 2000 The Association for the Study of Animal Behaviour.

One of the most important factors affecting the life history characteristics and biogeography of aquatic insects is temperature (Sweeney, 1984). Insects are poikilothermic (cold-blooded) animals whose metabolism, rate and magnitude of growth, development, and overall behavioral activities respond significantly to thermal change on a diel, seasonal, and annual basis (Ward and Stanford, 1982). Despite this sensitivity to temperature, most aquatic insect species can be found in aquatic habitats over a broad geographic area that includes a wide range of thermal regimes. Obviously, these aquatic insect species possess bioenergetic, developmental, and/or behavioral mechanisms that enable conspecific populations to survive and reproduce in very different environmental conditions.

The population genetic structure of two species of mayflies was studied using protein electrophoresis at eight locations in the headwater region of the Delaware River drainage basin. Study sites were located upstream and downstream from two large reservoirs. The working hypothesis was that little or no genetic differentiation occurs between conspecific populations within and among tributaries of the same drainage basin. A total of 24 and 28 loci were examined for Eurylophella verisimilis and Ephemerella subvaria respectively. Geographic differentiation was significant for three of nine polymorphic loci (average F ST =0.028) for Ep. subvaria when all populations were compared. Spatial variation in allele frequencies among populations of Ep. subvaria was substantially higher in the West Branch than in the East Branch of the Delaware River. The spatial cline of allele frequencies for certain loci of Ep. subvaria paralleled environmental gradients in the vicinity of the two reservoirs but no cause-ef...

We evaluated genetic variation in 40 populations (five species) of ephemerellid mayflies collected from streams in the Atlantic and Gulf drainages of eastern North America. The principal study species included Eurylophella funeralis (McDunnough), Eurylophella verisimilis (McDunnough), Ephemerella subvaria McDunnough, Ephemerella aurivillii (Bengtsson) and Ephemerella septentrionalis McDunnough. For the 40 populations, proportion of polymorphic loci averaged 22.3% (SEM = 2.0; range, 0–50%), with relatively low genie heterozygosity (ݲ ± SEM = 0.079 ± 0.006; range, 0–0.160). Nei's genetic distance ( D ) averaged 0.006 among conspecific mayfly populations and 1.434 among species. A genetic distance of 0.704 suggests that Eu. funeralis and Eu. verisimilis are distantly related congeners. Genetic distances among populations of the three principal Ephemerella species were even higher, with D values averaging >1.0. Genetic distance data for five additional Ephemerella species, Ephemerella dorothea Needham, Ephemerella excrucians Walsh, Ephemerella inconstans Traver, Ephemerella invaria (Walker), and Ephemerella rotunda Morgan, revealed that D values

The genus Eurylophella includes nine species from eastern North America, one from western North America, and three from Europe. We examined 18 polymorphic enzyme loci in 82 populations (>2,000 individuals) of Eurylophella from 40 localities in eastern North America, representing eight of the nine currently recognized eastern species. Our data suggest that Eurylophella is actually represented by at least 15 species in eastern North America, some of which cannot be resolved morphologically. Although intraspecific comparisons revealed significant geographic variation in allele frequencies, there were no fixed allelic differences between populations. In contrast, most interspecific comparisons were characterized by the lack of shared alleles at several loci. Thus, >99% of the individuals studied could be sorted to species by electromorph characteristics. The average Nei's genetic distance ( D ) was 0.02 (range = 0-0.12) between conspecific populations and 0.74 (range = 0.11-1.87) between species. A phenogram generated from D values shows five groups of species branching at D < 0.60: • E. verisimilis, E. verisimilis-A, E. verisimilis-B, E. verisimilis-C, E. bicolor, E. minimella, E. prudentalis, E. aestiva , and E. aestiva-A; • E. temporalis-A, E. temporalis-B , and E. temporalis-C; • E. sp. 1; • E. funeralis; and • E. lutulenta. Interspecific D values for Eurylophella were in the upper range of reported literature values for congeneric insect species, a fact that supports the recent elevation of Eurylophella from subgeneric to generic status. Expected heterozygosities ranged from 0.03 to 0.21; those of most species averaged 0.10-0.12.