An unusually large genome from an unusually large stonefly: a chromosome-length genome assembly for the giant salmonfly, Pteronarcys californica (Plecoptera: Pteronarcyidae).

Pteronarcys californica (Newport 1848) is commonly referred to as the giant salmonfly and is the largest species of stonefly (Insecta: Plecoptera) in the western United States. Historically, it was widespread and abundant in western rivers, but populations have experienced a substantial decline in the past few decades, becoming locally extirpated in numerous rivers in Utah, Colorado, and Montana. Although previous research has explored the ecological variables conducive to the survivability of populations of the giant salmonfly, a lack of genomic resources hampers exploration of how genetic variation is spread across extant populations. To accelerate research on this imperiled species, we present a de novo chromosomal-length genome assembly of P. californica generated from PacBio HiFi sequencing and Hi-C chromosome conformation capture. Our assembly includes 14 predicted pseudo chromosomes and 98.8% of Insecta universal core orthologs. At 2.40 gigabases, the P. californica assembly is the largest of available stonefly assemblies, highlighting at least 9.5-fold variation in assembly size across the order. Repetitive elements (REs) account for much of the genome size increase in P. californica relative to other stonefly species, with the content of Class I retroelements alone exceeding the entire assembly size of all but two other species studied. We also observed preliminary suborder-specific trends in genome size that merit testing with more robust taxon sampling.

Mitochondrial genomes of Nemourinae species (Plecoptera: Nemouridae) and the phylogenetic implications.

Currently, the classification system of 2 subfamilies within Nemouridae has been widely accepted. However, monophyly of 2 subfamilies has not been well supported by molecular evidence. To date, only mitogenomes from genus Nemoura of the subfamily Nemourinae were used in previous phylogenetic studies and produced conflicting results with morphological studies. Herein, we analyzed mitogenomes of 3 Nemourinae species to reveal their mitogenomic characteristics and to examine genus-level classification among Nemouridae. In this study, the genome organization of 3 mitogenomes is highly conserved in gene order, nucleotide composition, codon usage, and amino acid composition. In 3 Nemourinae species, there is a high variation in nucleotide diversity among the 13 protein-coding genes (PCGs). The Ka/Ks values for all PCGs were far lower than 1, indicating that these genes were evolving under purifying selection. The phylogenetic analyses highly support Nemurella as the sister group to Ostrocerca. Meanwhile, Nemoura is recovered as the sister group of Malenka; they are grouped with other Amphinemurinae and emerged from a paraphyletic Nemourinae. More molecular data from different taxonomic groups are needed to understand stoneflies phylogeny and evolution.

ebony underpins Batesian mimicry in melanic stoneflies.

The evolution of Batesian mimicry - whereby harmless species avoid predation through their resemblance to harmful species - has long intrigued biologists. In rare cases, Batesian mimicry is linked to intraspecific colour variation, in which only some individuals within a population resemble a noxious 'model'. Here, we assess intraspecific colour variation within a widespread New Zealand stonefly, wherein highly melanized individuals of Zelandoperla closely resemble a chemically defended aposematic stonefly, Austroperla cyrene. We assess convergence in the colour pattern of these two species, compare their relative palatability to predators, and use genome-wide association mapping to assess the genetic basis of this resemblance. Our analysis reveals that melanized Zelandoperla overlap significantly with Austroperla in colour space but are significantly more palatable to predators, implying that they are indeed Batesian mimics. Analysis of 194,773 genome-wide SNPs reveals an outlier locus (ebony) strongly differentiating melanic versus non-melanic Zelandoperla. Genotyping of 338 specimens from a single Zelandoperla population indicates that ebony explains nearly 70% of the observed variance in melanism. As ebony has a well-documented role in insect melanin biosynthesis, our findings indicate this locus has a conserved function across deeply divergent hexapod lineages. Distributional records suggest a link between the occurrence of melanic Zelandoperla and the forested ecosystems where the model Austroperla is abundant, suggesting the potential for adaptive shifts in this system underpinned by environmental change.

Spatial and phylogenetic structure of Alpine stonefly assemblages across seven habitats using DNA-species.

Stream ecosystems are spatially heterogeneous, with many different habitat patches distributed within a small area. The influence of this heterogeneity on the biodiversity of benthic insect communities is well documented; however, studies of the role of habitat heterogeneity in species coexistence and assembly remain limited. Here, we investigated how habitat heterogeneity influences spatial structure (beta biodiversity) and phylogenetic structure (evolutionary processes) of benthic stonefly (Plecoptera, Insecta) communities. We sampled 20 sites along two Alpine rivers, including seven habitats in four different reaches (headwaters, meandering, bar-braided floodplain, and lowland spring-fed). We identified 21 morphological species and delineated 52 DNA-species based on sequences from mitochondrial cox1 and nuclear ITS markers. Using DNA-species, we first analysed the patterns of variation in richness, diversity, and assemblage composition by quantifing the contribution of each reach and habitat to the overall DNA-species diversity using an additive partition analysis and distance-based redundancy analysis. Using gene-tree phylogenies, we assessed whether environmental filtering could lead to the co-occurrence of DNA-species using a two-step analysis to detect a phylogenetic signal. All four reaches significantly contributed to DNA-species richness, with the meandering reach having the highest contribution. Habitats had an effect on DNA-species diversity, where glide, riffle and, pool influenced the spatial structure of stonefly assemblage possibly due to the high habitat heterogeneity. Among the habitats, the pool showed significant phylogenetic clustering, suggesting high levels of evolutionary adaptation and strong habitat filtering. This assemblage structure may be caused by long-term stability of the habitat and the similar requirements for co-occurring species. Our study shows the importance of different habitats for the spatial and phylogenetic structure of stonefly assemblage and sheds light on the habitat-specific diversity that may help improve conservation practices.

Hypothesised life cycle adaptation of stonefly (Plecoptera) in response to increased water temperatures.

Water temperature is the most important and critical factor for freshwater invertebrates and it fluctuates with an increase in air temperature. In this study, the effect of water temperature on egg development was clarified in Stavsolus japonicus, and the response to climate change in stoneflies with long egg periods were considered. Water temperatures prior to 43 days before hatching likely do not affect egg development in Stavsolus japonicus. Instead, they use egg diapause as an adaptive strategy to survive hot summer conditions. Increased water temperatures may cause migration to higher elevations for stoneflies that have lower adaptability in their egg development period, with populations eventually becoming stranded where no higher elevation or cooler habitat is available. Species extinction is expected to increase with increasing temperatures, leading to reduced biodiversity in many ecosystems. Indirect effects of water warming on maturation and reproduction may lead to substantial reductions in benthic invertebrate populations.

Mining and urbanization affect river chemical water quality and macroinvertebrate communities in the upper Selenga River Basin, Mongolia.

Mongolia is a country with a quickly growing economy mainly based on mining of gold, copper, coal, and other minerals. Mining, urbanization, and agriculture impact the water quality in the upper Selenga River Basin in northern Mongolia, which is the center of the Mongolian economy. Previous measurements of pollution loads were alarming, but restricted to chemical measurements. Here, for the first time, we combine freshwater biomonitoring and laboratory water quality data across a broad gradient of water quality and land use intensity. We track the effects of different types of pollution on aquatic invertebrates and test their use as bioindicators. We collected water samples, environmental parameters, and macroinvertebrates at 36 sampling sites at the rivers of Tuul, Kharaa, and Orkhon and their tributaries Sugnugur, Boroo, Sharyn Gol, Gatsuurt, and Yeröö. PCA of catchment water quality distinguished three groups of pollutants prevalent at the sites: (1) nutrients, (2) saline components (Cl-, Na +, Mg2+, SO42-, Ca2+) and mining by-products (B, Sr, U, Mo), and (3) (heavy) metals, which often exceeded regulatory standards. We recorded a total of 59 macroinvertebrate taxa belonging to 32 families in seven insect orders plus Amphipoda and Gastropoda. Species diversity declined with higher impact. Five environmental factors structured macroinvertebrate community composition in RDA: elevation of sample location, site total nitrogen, dissolved oxygen, electrical conductivity, and water chemistry. We conclude that macroinvertebrate communities are an appropriate and inexpensive tool for monitoring water quality in Mongolia and suggest government action to establish a long-term monitoring program.

Genomic signatures of parallel alpine adaptation in recently evolved flightless insects.

Natural selection along elevational gradients has potential to drive predictable adaptations across distinct lineages, but the extent of such repeated evolution remains poorly studied for many widespread alpine taxa. We present parallel genomic analyses of two recently evolved flightless alpine insect lineages to test for molecular signatures of repeated alpine adaptation. Specifically, we compare low-elevation vs. alpine stonefly ecotypes from parallel stream populations in which flightless upland ecotypes have been independently derived. We map 67,922 polymorphic genetic markers, generated across 176 Zelandoperla fenestrata specimens from two independent alpine stream populations in New Zealand's Rock and Pillar Range, to a newly developed plecopteran reference genome. Genome-wide scans revealed 31 regions with outlier single nucleotide polymorphisms (SNPs) differentiating lowland vs. alpine ecotypes in Lug Creek, and 37 regions with outliers differentiating ecotypes in Six Mile Creek. Of these regions, 13% (8/60) yielded outlier SNPs across both within-stream ecotype comparisons, implying comparable genomic shifts contribute to this repeated alpine adaptation. Candidate genes closely linked to repeated outlier regions include several with documented roles in insect wing-development (e.g., dishevelled), suggesting that they may contribute to repeated alpine wing reduction. Additional candidate genes have been shown to influence insect fecundity (e.g., ovo) and lifespan (e.g., Mrp4), implying that they might contribute to life history differentiation between upland and lowland ecotypes. Additional outlier genes have potential roles in the evolution of reproductive isolation among ecotypes (hedgehog and Desaturase 1). These results demonstrate how replicated outlier tests across independent lineages can potentially contribute to the discovery of genes underpinning repeated adaptation.

Anthropogenic evolution in an insect wing polymorphism following widespread deforestation.

Anthropogenic environmental change can underpin major shifts in natural selective regimes, and can thus alter the evolutionary trajectories of wild populations. However, little is known about the evolutionary impacts of deforestation-one of the most pervasive human-driven changes to terrestrial ecosystems globally. Absence of forest cover (i.e. exposure) has been suggested to play a role in selecting for insect flightlessness in montane ecosystems. Here, we capitalize on human-driven variation in alpine treeline elevation in New Zealand to test whether anthropogenic deforestation has caused shifts in the distributions of flight-capable and flightless phenotypes in a wing-polymorphic lineage of stoneflies from the Zelandoperla fenestrata species complex. Transect sampling revealed sharp transitions from flight-capable to flightless populations with increasing elevation. However, these phenotypic transitions were consistently delineated by the elevation of local treelines, rather than by absolute elevation, providing a novel example of human-driven evolution in response to recent deforestation. The inferred rapid shifts to flightlessness in newly deforested regions have implications for the evolution and conservation of invertebrate biodiversity.

Mountain stoneflies may tolerate warming streams: Evidence from organismal physiology and gene expression.

Rapid glacier recession is altering the physical conditions of headwater streams. Stream temperatures are predicted to rise and become increasingly variable, putting entire meltwater-associated biological communities at risk of extinction. Thus, there is a pressing need to understand how thermal stress affects mountain stream insects, particularly where glaciers are likely to vanish on contemporary timescales. In this study, we measured the critical thermal maximum (CTMAX ) of stonefly nymphs representing multiple species and a range of thermal regimes in the high Rocky Mountains, USA. We then collected RNA-sequencing data to assess how organismal thermal stress translated to the cellular level. Our focal species included the meltwater stonefly, Lednia tumana, which was recently listed under the U.S. Endangered Species Act due to climate-induced habitat loss. For all study species, critical thermal maxima (CTMAX  > 20°C) far exceeded the stream temperatures mountain stoneflies experience (<10°C). Moreover, while evidence for a cellular stress response was present, we also observed constitutive expression of genes encoding proteins known to underlie thermal stress (i.e., heat shock proteins) even at low temperatures that reflected natural conditions. We show that high-elevation aquatic insects may not be physiologically threatened by short-term exposure to warm temperatures and that longer-term physiological responses or biotic factors (e.g., competition) may better explain their extreme distributions.

Tolerance of aquifer stoneflies to repeated hypoxia exposure and oxygen dynamics in an alluvial aquifer.

Aquatic insects cope with hypoxia and anoxia using a variety of behavioral and physiological responses. Most stoneflies (Plecoptera) occur in highly oxygenated surface waters, but some species live underground in alluvial aquifers containing heterogeneous oxygen concentrations. Aquifer stoneflies appear to be supported by methane-derived food resources, which they may exploit using anoxia-resistant behaviors. We documented dissolved oxygen dynamics and collected stoneflies over 5 years in floodplain wells of the Flathead River, Montana. Hypoxia regularly occurred in two wells, and nymphs of Paraperla frontalis were collected during hypoxic periods. We measured mass-specific metabolic rates (MSMRs) at different oxygen concentrations (12, 8, 6, 4, 2, 0.5 mg l-1, and during recovery) for 111 stonefly nymphs to determine whether aquifer and benthic taxa differed in hypoxia tolerance. Metabolic rates of aquifer taxa were similar across oxygen concentrations spanning 2 to 12 mg l-1 (P>0.437), but the MSMRs of benthic taxa dropped significantly with declining oxygen (P<0.0001; 2.9-times lower at 2 vs. 12 mg l-1). Aquifer taxa tolerated short-term repeated exposure to extreme hypoxia surprisingly well (100% survival), but repeated longer-term (>12 h) exposures resulted in lower survival (38-91%) and lower MSMRs during recovery. Our work suggests that aquifer stoneflies have evolved a remarkable set of behavioral and physiological adaptations that allow them to exploit the unique food resources available in hypoxic zones. These adaptations help to explain how large-bodied consumers might thrive in the underground aquifers of diverse and productive river floodplains.

Complex and nonlinear climate-driven changes in freshwater insect communities over 42 years.

The ongoing biodiversity crisis becomes evident in the widely observed decline in abundance and diversity of species, profound changes in community structure, and shifts in species' phenology. Insects are among the most affected groups, with documented decreases in abundance up to 76% in the last 25-30 years in some terrestrial ecosystems. Identifying the underlying drivers is a major obstacle as most ecosystems are affected by multiple stressors simultaneously and in situ measurements of environmental variables are often missing. In our study, we investigated a headwater stream belonging to the most common stream type in Germany located in a nature reserve with no major anthropogenic impacts except climate change. We used the most comprehensive quantitative long-term data set on aquatic insects available, which includes weekly measurements of species-level insect abundance, daily water temperature and stream discharge as well as measurements of additional physicochemical variables for a 42-year period (1969-2010). Overall, water temperature increased by 1.88 °C and discharge patterns changed significantly. These changes were accompanied by an 81.6% decline in insect abundance, but an increase in richness (+8.5%), Shannon diversity (+22.7%), evenness (+22.4%), and interannual turnover (+34%). Moreover, the community's trophic structure and phenology changed: the duration of emergence increased by 15.2 days, whereas the peak of emergence moved 13.4 days earlier. Additionally, we observed short-term fluctuations (<5 years) in almost all metrics as well as complex and nonlinear responses of the community toward climate change that would have been missed by simply using snapshot data or shorter time series. Our results indicate that climate change has already altered biotic communities severely even in protected areas, where no other interacting stressors (pollution, habitat fragmentation, etc.) are present. This is a striking example of the scientific value of comprehensive long-term data in capturing the complex responses of communities toward climate change.

Cambios Complejos y No Lineales Causados por el Clima en Comunidades de Insectos de Agua Dulce durante 42 Años Resumen La continua crisis de la biodiversidad se vuelve evidente en la ampliamente documentada declinación en la abundancia y diversidad de especies, cambios profundos en la estructura de las comunidades y modificaciones en la fenología de las especies. Los insectos se encuentran entre los grupos más afectados; se han documentado reducciones en la abundancia de hasta el 76% en los últimos 25-30 años en algunos ecosistemas terrestres. La identificación de los causantes subyacentes es un gran obstáculo porque la mayoría de los ecosistemas están afectados por varios factores estresantes simultáneamente y con frecuencia faltan las medidas in situ de las variables ambientales. Investigamos un flujo naciente perteneciente al tipo de arroyo más común en Alemania ubicado dentro de una reserva natural sin impactos antropogénicos importantes a excepción del cambio climático. Usamos el conjunto más completo disponible de datos cuantitativos de largo plazo para insectos acuáticos que incluye las medidas semanales de abundancia de insectos a nivel especie, las medidas diarias de la temperatura del agua y la descarga del flujo y las medidas de variables físico-químicas adicionales durante 42 años (1969 - 2010). En general, la temperatura del agua incrementó 1.88°C y los patrones de descarga cambiaron significativamente. Estos cambios estuvieron acompañados por una declinación del 81.6% en la abundancia de insectos, pero también de un incremento en la riqueza (+8.5%), la diversidad Shannon (+22.7%), la uniformidad (+22.4%) y la renovación interanual (+34%). Además, la estructura trófica y la fenología de la comunidad cambiaron: la duración del surgimiento incrementó en 15.2 días y el pico del surgimiento ocurrió con 13.4 días de anticipación. Observamos fluctuaciones a corto plazo (<5 años) en casi todas las medidas, así como respuestas complejas y no lineales de la comunidad hacia el cambio climático que podrían haber sido ignoradas si sólo se usaran datos instantáneos o series de tiempo más cortas. Nuestros resultados indican que el cambio climático ya ha alterado seriamente a las comunidades bióticas, incluso en áreas protegidas en las que no están presentes otros factores estresantes en interacción (contaminación, fragmentación del hábitat, etc.). Ésto es un ejemplo notable del valor científico que tienen los datos completos de escalas de tiempo a largo plazo para la captura de las respuestas complejas de las comunidades ante el cambio climático.

Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

The phylogeny and evolutionary timescale of stoneflies (Insecta: Plecoptera) inferred from mitochondrial genomes.

Phylogenetic analysis based on mitochondrial genomic data from 25 stonefly species recovered a well-supported tree resolving higher-level relationships within Plecoptera (stoneflies). The monophyly of both currently recognized suborders was strongly supported, concordant with previous molecular analyses of Plecoptera. The southern hemisphere suborder Antarctoperlaria formed two clades: Eustheniidae + Diamphipnoidae and Austroperlidae + Gripopterygidae; consistent with relationships proposed based on morphology. The largely northern hemisphere suborder Arctoperlaria also divided into two groups, Euholognatha and Systellognatha, each composed of the five families traditionally assigned to each infraorder (the placement Scopuridae by mt genome data remains untested at this time). Within Euholognatha, strong support for the clade Nemouridae + Notonemouridae confirmed the northern origin of the currently southern hemisphere restricted Notonemouridae. Other family level relationships within the Arctoperlaria differ from those recovered by previous morphology and molecular based analyses. A fossil-calibrated divergence estimation suggests the formation of two suborders dates back to the Jurassic (181 Ma), with subsequent diversification of most stonefly families during the Cretaceous. This result confirms the hypothesis that initial divergence between the suborders was driven by the breakup of the supercontinent Pangaea into Laurasia and Gondwanaland (commencing 200 Ma and complete by 150 Ma).

Complete Mitochondrial Genome of Suwallia teleckojensis (Plecoptera: Chloroperlidae) and Implications for the Higher Phylogeny of Stoneflies.

Stoneflies comprise an ancient group of insects, but the phylogenetic position of Plecoptera and phylogenetic relations within Plecoptera have long been controversial, and more molecular data is required to reconstruct precise phylogeny. Herein, we present the complete mitogenome of a stonefly, Suwallia teleckojensis, which is 16146 bp in length and consists of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and a control region (CR). Most PCGs initiate with the standard start codon ATN. However, ND5 and ND1 started with GTG and TTG. Typical termination codons TAA and TAG were found in eleven PCGs, and the remaining two PCGs (COII and ND5) have incomplete termination codons. All transfer RNA genes (tRNAs) have the classic cloverleaf secondary structures, with the exception of tRNASer(AGN), which lacks the dihydrouridine (DHU) arm. Secondary structures of the two ribosomal RNAs were shown referring to previous models. A large tandem repeat region, two potential stem-loop (SL) structures, Poly N structure (2 poly-A, 1 poly-T and 1 poly-C), and four conserved sequence blocks (CSBs) were detected in the control region. Finally, both maximum likelihood (ML) and Bayesian inference (BI) analyses suggested that the Capniidae was monophyletic, and the other five stonefly families form a monophyletic group. In this study, S. teleckojensis was closely related to Sweltsa longistyla, and Chloroperlidae and Perlidae were herein supported to be a sister group.

Climate-induced glacier and snow loss imperils alpine stream insects.

Climate warming is causing rapid loss of glaciers and snowpack in mountainous regions worldwide. These changes are predicted to negatively impact the habitats of many range-restricted species, particularly endemic, mountaintop species dependent on the unique thermal and hydrologic conditions found only in glacier-fed and snow melt-driven alpine streams. Although progress has been made, existing understanding of the status, distribution, and ecology of alpine aquatic species, particularly in North America, is lacking, thereby hindering conservation and management programs. Two aquatic insects - the meltwater stonefly (Lednia tumana) and the glacier stonefly (Zapada glacier) - were recently proposed for listing under the U.S. Endangered Species Act due to climate-change-induced habitat loss. Using a large dataset (272 streams, 482 total sites) with high-resolution climate and habitat information, we describe the distribution, status, and key environmental features that limit L. tumana and Z. glacier across the northern Rocky Mountains. Lednia tumana was detected in 113 streams (175 sites) within Glacier National Park (GNP) and surrounding areas. The probability of L. tumana occurrence increased with cold stream temperatures and close proximity to glaciers and permanent snowfields. Similarly, densities of L. tumana declined with increasing distance from stream source. Zapada glacier was only detected in 10 streams (24 sites), six in GNP and four in mountain ranges up to ~600 km southwest. Our results show that both L. tumana and Z. glacier inhabit an extremely narrow distribution, restricted to short sections of cold, alpine streams often below glaciers predicted to disappear over the next two decades. Climate warming-induced glacier and snow loss clearly imperils the persistence of L. tumana and Z. glacier throughout their ranges, highlighting the role of mountaintop aquatic invertebrates as sentinels of climate change in mid-latitude regions.

Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies.

First Mitochondrial Genome from Nemouridae (Plecoptera) Reveals Novel Features of the Elongated Control Region and Phylogenetic Implications.

The complete mitochondrial genome (mitogenome) of Nemoura nankinensis (Plecoptera: Nemouridae) was sequenced as the first reported mitogenome from the family Nemouridae. The N. nankinensis mitogenome was the longest (16,602 bp) among reported plecopteran mitogenomes, and it contains 37 genes including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Most PCGs used standard ATN as start codons, and TAN as termination codons. All tRNA genes of N. nankinensis could fold into the cloverleaf secondary structures except for trnSer (AGN), whose dihydrouridine (DHU) arm was reduced to a small loop. There was also a large non-coding region (control region, CR) in the N. nankinensis mitogenome. The 1751 bp CR was the longest and had the highest A+T content (81.8%) among stoneflies. A large tandem repeat region, five potential stem-loop (SL) structures, four tRNA-like structures and four conserved sequence blocks (CSBs) were detected in the elongated CR. The presence of these tRNA-like structures in the CR has never been reported in other plecopteran mitogenomes. These novel features of the elongated CR in N. nankinensis may have functions associated with the process of replication and transcription. Finally, phylogenetic reconstruction suggested that Nemouridae was the sister-group of Capniidae.

A time-calibrated phylogeny of southern hemisphere stoneflies: Testing for Gondwanan origins.

For more than two centuries biogeographers have attempted to explain why terrestrial or freshwater lineages have geographic distributions broken by oceans, with these disjunct distributions either attributed to vicariance associated with Gondwanan fragmentation or trans-oceanic dispersal. Stoneflies (order: Plecoptera) are a widespread order of freshwater insects whose poor dispersal ability and intolerance for salt water make them ideal candidates for Gondwanan relicts - taxa whose distribution can be explained by vicariant isolation driven by the breakup of Gondwana. Here we reconstruct the phylogenetic relationships among southern hemisphere stoneflies (5 families; 86 genera) using 2864bp of mitochondrial (COI) and nuclear (18S, H3) DNA, with a calibrated relaxed molecular clock used to estimate the chronology of diversification. Our analysis suggests that largely antitropical stonefly sub-orders, Arctoperlaria (northern hemisphere) and Antarctoperlaria (southern hemisphere), were formed approximately 121Ma (95% prior probability distribution 107-143Ma), which may reflect the vicariant rifting of the supercontinent Pangaea. Subsequently, we infer that a single Arctoperlaria lineage has dispersed into southern hemisphere 76Ma (95% range 65-98Ma). The majority of divergences between South American and Australian stonefly lineages appear to coincide with the opening of Drake Passage around 40Ma, suggesting vicariant isolation of these landmasses may be responsible for these biogeographic disjunctions. In contrast, divergences between New Zealand lineages and their sister taxa appear to post-date vicariant timeframes, implying more recent dispersal events.

Examining spatial patterns in polycyclic aromatic compounds measured in stream macroinvertebrates near a small subarctic oil and gas operation.

The Cameron River runs through a small, remote petrochemical development in the Cameron Hills (Northwest Territories, Canada). In order to evaluate the exposure of aquatic biota to contaminants from oil and gas activities, we measured polycyclic aromatic compounds (PACs) in macroinvertebrates collected from sites and tributaries along the Cameron River, including upstream and downstream of the development, and sites located near drilled wells (developed). Macroinvertebrate tissue PAC burdens ranged from 0.2-2.8 µg g(-1) lipid for unsubstituted compounds, and from 4.2-63.2 µg g(-1) lipid for alkylated compounds, relatively low compared to similar studies from more industrialized regions in North America. There was no significant difference in tissue PAC burdens between upstream, downstream, or developed sites (p = 0.12), although alkyl PACs in five out of seven developed sites were higher than the regional average. Petrogenic PACs were dominant in most samples, including alkyl fluorines, alkyl phenanthrene/anthracenes, and alkyl dibenzothiophenes. Minimal changes in PAC composition in macroinvertebrate tissues were detected along the Cameron River, with the exception of the two sites furthest downstream that had high concentrations of C3-C4 naphthalene. Overall, our results suggest that oil and gas development in the Cameron Hills has not resulted in substantial increases in PAC bioaccumulation in stream macroinvertebrates, although the potential that alkyl naphthalenes are being transported downstream from the development warrants further attention.

The decapod red pigment-concentrating hormone (Panbo-RPCH) is the first identified neuropeptide of the order Plecoptera and is interpreted as homoplastic character state.

This paper presents the first neuropeptide structure, identified by mass spectrometry, in two species of Plectoptera (stoneflies) and in one species of the coleopteran family Lycidae. In all three species, the octapeptide Panbo-RPCH (first identified in Pandalus borealis as a red pigment-concentrating hormone: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp amide) is present. A review of the literature available on invertebrate neuropeptides that are identified or predicted from expressed sequence tags, transcriptome shotgun assemblies, and from fully sequenced genomes, show that Panbo-RPCH is found in Malacostraca (Crustacea) and certain hemipteran Heteroptera (Insecta). To date, Panbo-RPCH has not been shown present in non-Malacostracan crustaceans, nor in basal taxa of the Insecta (Archaeognatha, Zygentoma, Ephemeroptera, Odonata). The present data adds to knowledge on the distribution of Panbo-RPCH, and when taking into account the most accepted, current phylogenetics of the Crustacea-Hexapoda relationship, this distribution of Panbo-RPCH in Malacostraca, Plecoptera, some hemipteran Heteroptera and in Coleoptera (Lycidae) can best be explained by homoplasy, implying parallel evolution.