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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 ...

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.

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.

Temperature dictates the performance of aquatic ectotherms. However, the physiological and biochemical processes that drive thermally-mediated life history patterns (and limits) remain poorly understood because they are rarely studied simultaneously. In our previous work, we have established life history outcomes (e.g. survivorship, development time, growth rates and fitness) in mayflies (Neocloeon triangulifer) reared at static temperatures ranging from 14°C - 30°C at 2°C intervals. In this study, we conducted biochemical measurements (RT-qPCR of select genes and targeted, quantitative metabolomic profiling) on N. triangulifer mature larvae reared at temperatures associated with excellent survival and fitness (22-24°C), compromised survival and fitness (28°C), and chronic lethality (30°C -larvae survived for a few weeks but failed to emerge to adulthood). Patterns of gene expression were similar to those observed in acute ramping experiments reported previously: larvae reared at 30°C resulted in significant upregulation in the thermally responsive gene HEAT SHOCK PROTEIN 90 (HSP90) but no significant changes in hypoxia responsive genes (EGG LAYING DEFECTIVE 9 (EGL-9) and LACTATE DEHYDROGENASE (LDH)). Additionally, primers for genes associated with energy: INSULIN RECEPTOR (IR), mechanistic TARGET OF RAPAMYCIN (mTOR) and TREHALOSE 6 PHOSPHATE SYNTHASE (T6PS) were developed for this study. IR and mTOR were significantly upregulated while T6PS showed trend of downregulation in larvae reared at 30°C. Metabolomic profiles revealed general depletion of lipids and acylcarnitines in larvae exposed to chronic thermal stress, suggesting that larvae were energetically challenged despite continuous access to food. For example, concentrations of lysoPhosphatidylcholine (lysoPC) a C20:3 decreased as fitness decreased with increasing temperature (2.3 fold and 2.4 fold at 28 and 30°C relative to controls). Tissue concentrations of the biogenic amine histamine increased 2.1 and 3.1 fold with increasing temperature, and were strongly and negatively correlated with performance. Thus, both histamine and lysoPC a C20:3 are potential biomarkers of thermal stress. Taken together, our results primarily associate energetic challenge with thermally mediated fitness reduction in N. triangulifer.

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.

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%) ...

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).

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...

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.

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.

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...