Declines in an abundant aquatic insect, the burrowing mayfly, across major North American waterways.

Seasonal animal movement among disparate habitats is a fundamental mechanism by which energy, nutrients, and biomass are transported across ecotones. A dramatic example of such exchange is the annual emergence of mayfly swarms from freshwater benthic habitats, but their characterization at macroscales has remained impossible. We analyzed radar observations of mayfly emergence flights to quantify long-term changes in annual biomass transport along the Upper Mississippi River and Western Lake Erie Basin. A single emergence event can produce 87.9 billion mayflies, releasing 3,078.6 tons of biomass into the airspace over several hours, but in recent years, production across both waterways has declined by over 50%. As a primary prey source in aquatic and terrestrial ecosystems, these declines will impact higher trophic levels and environmental nutrient cycling.

Altitudinal and seasonal distribution of benthic macroinvertebrates in River Tons - a tributary of Yamuna River, Uttarakhand, India.

The main tributary of the Yamuna, the Tons River, exhibits altitudinal changes in its macroinvertebrate community's diversity, abundance, and composition. Between May 2019 and April 2021, the study was conducted in the upper section of the river. A total of 48 numbers of taxa from 34 families and ten orders were recorded during the investigation. At this elevation of 1150 to 1287 m, the two most predominant orders are Ephemeroptera (32.9%) and Trichoptera (29.5%). During the premonsoon season, they had the lowest macroinvertebrate density (250-290 individuals/m2), and the post-monsoon season had the highest density (600-640 individuals/m2). During the post-monsoon season, the maximum larval forms (60%) of various insect orders were predominant. The findings indicated that lower altitudes (1150-1232 m) have higher macroinvertebrate abundance than higher ones. The diversity of dominance is shallow at site-I (0.0738) and strong at the site-IV during the premonsoon season (0.03837). Taxa richness, as measured by the Margalef index (D), peaked in the spring season (January to March) at 6.9 and reached its lowest point (5.74) in the premonsoon season (April to May). Only 16 taxa were discovered in site-I and site-II, but 39 taxa were discovered at low altitudes (site-IV, 1100 m) (1277-1287 m). The Tons River contains a total of 12 and 13 genera, respectively, that belong to the orders Ephemeroptera and Trichoptera, according to qualitative study of the macroinvertebrates. The current study supports the use of macroinvertebrates as bioindicator species for monitoring biodiversity and assessing the health of ecosystems.

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.

Baetid abundances are a rapid indicator of thermal stress and riparian zone intactness.

Riparian zone vegetation plays an integral role in freshwater ecology, notably by buffering water temperatures, and in providing habitat for the adult stages of many aquatic species. We measured the contribution that riparian vegetation makes to temperature buffering, and how this affects the freshwater fauna, specifically using changes in abundances of baetid may flies for the Luvuvhu River catchment in South Africa. Water temperatures were compared for shaded versus un-shaded sites, and thermal stress between seasons was estimated using a cumulative probability model for the most widespread mayfly species, Dabulamanzia media. It is concluded that thermal stress due to losses in riparian shading could be detected using mayfly abundances in a regular monitoring programme.

The influence of the environment in the incorporation of copper and cadmium in scraper insects.

In regions with intense agricultural activity, increases in heavy metal concentrations in aquatic environments are common. Among the metals associated with agricultural activities, copper (Cu) and cadmium (Cd) have been found to directly affect aquatic trophic structures due to the ease of incorporation by primary producers and consuming organisms. Aquatic insects are predominantly found in streams, and their presence is determined by environmental characteristics and food availability. In this study, we seek to understand how the incorporation of Cu and Cd by scraper insects relates to their environment and food sources. We collected water, sediment, biofilm and scraper insect samples in streams with different intensities of agricultural activities in the drainage areas. The intensity of agricultural activities in the catchment area positively influenced the Cu and Cd concentrations in organisms and other aquatic compartments. The metals were readily incorporated by the biofilms. Although the functional characteristics are important to understand the functioning of ecosystems, in this study, we found that the physiological characteristics can be determinants in the concentrations of metals in aquatic insects.

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.

The First Establishment of "Hand-Pairing" Cross-Breeding Method for the Most Ancestral Wing Acquired Insect Group.

Insects are the most diverse organisms in the world and have been in existence since ca. 480 Ma; given this, they can provide profound insights into evolution. Among them, the order Ephemeroptera is one of the most basal clades of winged insects. This makes Ephemeroptera a significant key taxon in understanding the macro-evolution or the insect groundplan. In the development of biological evolutionary studies of this taxon, it is important to establish a technique for cross-breeding. Furthermore, the establishment of these techniques also makes a great contribution in the fields of micro-evolution. In a non-model taxon, the mayfly, subcultivation in the laboratory has been thus far considered impossible. With the exception of some parthenogenetic strains, it is extremely difficult to mate these insects in artificial environments. In this study, we established a successful artificial mating technique, i.e., a "hand-pairing" based cross-breeding method for mayflies. Furthermore, we also succeeded in clearly verifying by a genotyping method that the offspring reproduced by hand-pairing were in fact derived from the actual male and female which were used for hand-pairing. We established a reproductive experimental technique for hand-pairing of Dipteromimus tipuliformis and verified this technique by means genotyping. This technique could allow the artificial control of fertilization timing, and result in offspring which can be verified as to their status by means of genotyping. This achievement will be extremely important in the future for both the macro- and micro-evolutionary studies of insects.

Clarifying the phylogenetic relationships and taxonomy of Stenonema, Stenacron and Maccaffertium, three common eastern North American mayfly genera.

Stenonema, Stenacron, and Maccaffertium are three closely related genera of mayflies (Ephemeroptera:Heptageniidae) commonly found across North America. Due to their primarily aquatic life history and sensitivity to aquatic pollutants, these mayflies are often used as water quality indicators. However, there is little morphological variation within these genera, leading to difficulties in identification and rampant taxonomic confusion, limiting their utility as bioindicators. In an attempt to resolve the phylogenetic relationships of Stenonema, Stenacron, and Maccaffertium, and to clarify their higher-level classifications, we sequenced regions of two mitochondrial genes (cytochrome oxidase subunit 1 (cox1) and 16S ribosomal RNA (rrnl)) and two nuclear genes (Wingless (Wg) and histone H3) from 60 individuals representing most of the described species in these genera and included data from representatives of three heptageniid genera (Kageronia, Macdunnoa and Pseudiron) proposed in previous studies to be closely related to our focal taxa as well as two more distantly related heptageniid genera (Epeorus and Heptagenia) to root the phylogenies. Maximum likelihood and Bayesian analysis were conducted on single-gene and concatenated multi-gene data sets and species tree methods were utilized to resolve relationships. These analyses resolved Stenacron as a monophyletic group sister to a clade comprising Macdunnoa, Maccaffertium and Stenonema. Maccaffertium was found to be paraphyletic, with Stenonema femoratum resolved within Maccaffertium as sister to M. mexicanum. Many relationships remained unresolved or varied across analyses, making revision of the classification based on phylogenetic considerations challenging. To minimize confusion while naming clades and acknowledging uncertainty in our phylogenetic conclusions, we redefine Stenonema to include Maccaffertium and propose three subgenera-Stenonema, Maccaffertium and Lewisa- for key well-supported clades.

A biomechanical analysis of prognathous and orthognathous insect head capsules: evidence for a many-to-one mapping of form to function.

Insect head shapes are remarkably variable, but the influences of these changes on biomechanical performance are unclear. Among 'basal' winged insects, such as dragonflies, mayflies, earwigs and stoneflies, some of the most prominent anatomical changes are the general mouthpart orientation, eye size and the connection of the endoskeleton to the head. Here, we assess these variations as well as differing ridge and sclerite configurations using modern engineering methods including multibody dynamics modelling and finite element analysis in order to quantify and compare the influence of anatomical changes on strain in particular head regions and the whole head. We show that a range of peculiar structures such as the genal/subgenal, epistomal and circumocular areas are consistently highly loaded in all species, despite drastically differing morphologies in species with forward-projecting (prognathous) and downward-projecting (orthognathous) mouthparts. Sensitivity analyses show that the presence of eyes has a negligible influence on head capsule strain if a circumocular ridge is present. In contrast, the connection of the dorsal endoskeletal arms to the head capsule especially affects overall head loading in species with downward-projecting mouthparts. Analysis of the relative strains between species for each head region reveals that concerted changes in head substructures such as the subgenal area, the endoskeleton and the epistomal area lead to a consistent relative loading for the whole head capsule and vulnerable structures such as the eyes. It appears that biting-chewing loads are managed by a system of strengthening ridges on the head capsule irrespective of the general mouthpart and head orientation. Concerted changes in ridge and endoskeleton configuration might allow for more radical anatomical changes such as the general mouthpart orientation, which could be an explanation for the variability of this trait among insects. In an evolutionary context, many-to-one mapping of strain patterns onto a relatively similar overall head loading indeed could have fostered the dynamic diversification processes seen in insects.

Molecular phylogeny and timing of diversification in Alpine Rhithrogena (Ephemeroptera: Heptageniidae).

BACKGROUND: Larvae of the Holarctic mayfly genus Rhithrogena Eaton, 1881 (Ephemeroptera, Heptageniidae) are a diverse and abundant member of stream and river communities and are routinely used as bio-indicators of water quality. Rhithrogena is well diversified in the European Alps, with a number of locally endemic species, and several cryptic species have been recently detected. While several informal species groups are morphologically well defined, a lack of reliable characters for species identification considerably hampers their study. Their relationships, origin, timing of speciation and mechanisms promoting their diversification in the Alps are unknown. RESULTS: Here we present a species-level phylogeny of Rhithrogena in Europe using two mitochondrial and three nuclear gene regions. To improve sampling in a genus with many cryptic species, individuals were selected for analysis according to a recent DNA-based taxonomy rather than traditional nomenclature. A coalescent-based species tree and a reconstruction based on a supermatrix approach supported five of the species groups as monophyletic. A molecular clock, mapped on the most resolved phylogeny and calibrated using published mitochondrial evolution rates for insects, suggested an origin of Alpine Rhithrogena in the Oligocene/Miocene boundary. A diversification analysis that included simulation of missing species indicated a constant speciation rate over time, rather than any pronounced periods of rapid speciation. Ancestral state reconstructions provided evidence for downstream diversification in at least two species groups. CONCLUSIONS: Our species-level analyses of five gene regions provide clearer definitions of species groups within European Rhithrogena. A constant speciation rate over time suggests that the paleoclimatic fluctuations, including the Pleistocene glaciations, did not significantly influence the tempo of diversification of Alpine species. A downstream diversification trend in the hybrida and alpestris species groups supports a previously proposed headwater origin hypothesis for aquatic insects.

Cryptic species diversity reveals biogeographic support for the 'mountain passes are higher in the tropics' hypothesis.

The 'mountain passes are higher in the tropics' (MPHT) hypothesis posits that reduced climate variability at low latitudes should select for narrower thermal tolerances, lower dispersal and smaller elevational ranges compared with higher latitudes. These latitudinal differences could increase species richness at low latitudes, but that increase may be largely cryptic, because physiological and dispersal traits isolating populations might not correspond to morphological differences. Yet previous tests of the MPHT hypothesis have not addressed cryptic diversity. We use integrative taxonomy, combining morphology (6136 specimens) and DNA barcoding (1832 specimens) to compare the species richness, cryptic diversity and elevational ranges of mayflies (Ephemeroptera) in the Rocky Mountains (Colorado; approx. 40°N) and the Andes (Ecuador; approx. 0°). We find higher species richness and smaller elevational ranges in Ecuador than Colorado, but only after quantifying and accounting for cryptic diversity. The opposite pattern is found when comparing diversity based on morphology alone, underscoring the importance of uncovering cryptic species to understand global biodiversity patterns.

Automated identification of aquatic insects: A case study using deep learning and computer vision techniques.

Deep learning techniques have recently found application in biodiversity research. Mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddisflies (Trichoptera), often abbreviated as EPT, are frequently used for freshwater biomonitoring due to their large numbers and sensitivity to environmental changes. However, the morphological identification of EPT species is a challenging but fundamental task. Morphological identification of these freshwater insects is therefore not only extremely time-consuming and costly, but also often leads to misjudgments or generates datasets with low taxonomic resolution. Here, we investigated the application of deep learning to increase the efficiency and taxonomic resolution of biomonitoring programs. Our database contains 90 EPT taxa (genus or species level), with the number of images per category ranging from 21 to 300 (16,650 in total). Upon completion of training, a CNN (Convolutional Neural Network) model was created, capable of automatically classifying these taxa into their appropriate taxonomic categories with an accuracy of 98.7 %. Our model achieved a perfect classification rate of 100 % for 68 of the taxa in our dataset. We achieved noteworthy classification accuracy with morphologically closely related taxa within the training data (e.g., species of the genus Baetis, Hydropsyche, Perla). Gradient-weighted Class Activation Mapping (Grad-CAM) visualized the morphological features responsible for the classification of the treated species in the CNN models. Within Ephemeroptera, the head was the most important feature, while the thorax and abdomen were equally important for the classification of Plecoptera taxa. For the order Trichoptera, the head and thorax were almost equally important. Our database is recognized as the most extensive aquatic insect database, notably distinguished by its wealth of included categories (taxa). Our approach can help solve long-standing challenges in biodiversity research and address pressing issues in monitoring programs by saving time in sample identification.

Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries.

Mayflies (Ephemeroptera) are among the crucial water and habitat quality bioindicators. However, despite their intensive long-term use in various studies, more reliable mayfly DNA barcode data have been produced in a negligible number of countries, and only ~40% of European species had been barcoded with less than 50% of families covered. Despite being carried out in a small area, our study presents the second-most species-rich DNA reference library of mayflies from Europe and the first comprehensive view from an important biodiversity hotspot such as the Western Carpathians. Within 1153 sequences, 76 morphologically determined species were recorded and added to the Barcode of Life Data System (BOLD) database. All obtained sequences were assigned to 97 BINs, 11 of which were unique and three represented species never barcoded before. Sequences of 16 species with high intraspecific variability were divided into 40 BINs, confirming the presence of cryptic lineages. Due to the low interspecific divergence and the non-existing barcoding gap, sequences of six species were assigned to three shared BINs. Delimitation analyses resulted in 79 and 107 putative species respectively. Bayesian and maximum-likelihood phylogenies confirmed the monophyly of almost all species and complexes of cryptic taxa and proved that DNA barcoding distinguishes almost all studied mayfly species. We have shown that it is still sufficient to thoroughly investigate the fauna of a small but geographically important area to enrich global databases greatly. In particular, the insights gained here transcend the local context and may have broader implications for advancing barcoding efforts.

Four decades of region- and species-specific trends in lowland stream Ephemeroptera abundance.

Lowland stream ecosystems are under threat from climate change, industrialization, urbanization, and intensive agriculture. Since the 1980s, improvements in water quality have led to an increase in lowland stream biodiversity. Despite restoration efforts, however, further recovery is often hampered by the presence of region-specific (combinations of) stressors, and species-specific stressor responses. Identification of these stressors may not be achieved by the analysis of abundance data over large areas for entire communities or species assemblages. Therefore, our study introduces an alternative in-depth approach, selecting Ephemeroptera as a model organism group and analyzing 41 years of species abundance data across distinct geographical regions. Our findings revealed that 15 Ephemeroptera species had already disappeared before 1985, emphasizing the importance of evaluating an extended historical period when analyzing biodiversity trends. While biodiversity was generally characterized by an initial recovery that stagnated over time, the analysis of the past 41 years of Ephemeroptera abundance data revealed strong differences in species' abundance trends between periods, regions, and species. Certain species were likely to have benefitted from local restoration measures in specific geographical regions, while others may have declined due to the presence of region-specific stressors. Our approach underscores the importance of studying the development of region- and species-specific stream biodiversity trends over time to aid the selection of the appropriate restoration measures to recover lowland stream biodiversity.

The genome sequence of the Large Brook Dun, Ecdyonurus torrentis (Kimmins, 1942).

We present a genome assembly from an individual female Ecdyonurus torrentis (the Large Brook Dun; Arthropoda; Insecta; Ephemeroptera; Heptageniidae). The genome sequence is 503.2 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 15.69 kilobases in length.

The genome sequence of the Northern Summer Mayfly, Siphlonurus alternatus (Say, 1824).

We present a genome assembly from an individual male Siphlonurus alternatus (the Northern Summer Mayfly; Arthropoda; Insecta; Ephemeroptera; Siphlonuridae). The genome sequence is 455.8 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 19.36 kilobases in length.

The Impact of Hydromorphological Alterations on Mayfly Assemblages of a Mid-Sized Lowland River in South-Eastern Europe.

Historically, rivers have been anthropogenically modified for different purposes worldwide (e.g., flood control, drinking water abstraction, and land drainage). Although the majority of European lowland rivers are under the impact of multiple stressors (water quality, hydromorphological alterations, land-use), the consequences of these influences on aquatic macroinvertebrates, including mayflies, have not been sufficiently studied. Therefore, with the aim of providing additional data on the response of mayflies to anthropogenic disturbances in riverine habitats, we studied mayfly assemblages and their relationship to environmental factors along a mid-sized lowland river in Croatia. No significant differences in mayfly species richness and their functional traits were observed among the three habitat groups with different levels of hydromorphological alterations (near-natural, moderately altered, and severely altered habitats). This could be related to the river's connection to the numerous tributaries, and the variety of available microhabitats along the studied system, despite the existing hydromorphological pressures. A stronger relationship was found between mayflies and the physico-chemical water characteristics and land-use. Water temperature, water velocity, oxygen content, and nutrient (e.g., nitrogen, phosphorous) concentrations related to agricultural and urban land-use were found to be the most important factors shaping mayfly assemblages in the studied lotic lowland system. These results can contribute to the planning of management and conservation activities for lowland rivers and their biota according to the requirements of the European Water Framework Directive.

Seasonal modifications of longitudinal distribution patterns within a stream: Interspecific interactions in the niche overlap zones of two Ephemera mayflies.

Niche differentiation between closely related species leads to differentiation of their habitats. Segregation based on slight differences in environmental factors, that is niche differentiation on the microhabitat scale, allows more species to inhabit a certain geographic space. Therefore, such fine scale niche differentiation is an important factor in the support of species diversity. In addition, niche differentiation on the microhabitat scale and/or the differentiation of breeding seasons can be considered typical mechanisms that facilitate multispecies' co-existence. In this study, sister species (Commonly, Ephemera japonica inhabit at upstream region and Ephemera strigata inhabit at middle stream region), which often coexist in the upper to middle reaches of river systems of the Japanese Islands, were targeted and the following aspects were investigated. First, differences in habitat preference and interspecific differences in flow distribution patterns on a geographically fine scale were tracked in detail. Subsequently, the temporal transitions of their distribution patterns were investigated in detail and seasonal changes were investigated. Finally, we thoroughly investigated the disappearance of nymphs of each species from the river due to emergence affected the distribution of each species (by conducting daily emergence surveys). Combining results of these multiple studies also suggested that there may be spatiotemporal interspecial interaction between these two species within/around their overlapping regions. Traditionally, the longitudinal distribution pattern of these two Ephemera mayflies has been thought to be established based on a difference in habitat preferences, but this study revealed that the interspecific interaction between the two species also plays an important role. This study provides new insights into species diversity and distribution pattern formation in river-dwelling species.

Characterizing Hox genes in mayflies (Ephemeroptera), with Hexagenia limbata as a new mayfly model.

BACKGROUND: Hox genes are key regulators of appendage development in the insect body plan. The body plan of mayfly (Ephemeroptera) nymphs differs due to the presence of abdominal appendages called gills. Despite mayflies' phylogenetic position in Paleoptera and novel morphology amongst insects, little is known of their developmental genetics, such as the appendage-regulating Hox genes. To address this issue we present an annotated, early instar transcriptome and embryonic expression profiles for Antennapedia, Ultrabithorax, and Abdominal A proteins in the mayfly Hexagenia limbata, identify putative Hox protein sequences in the mayflies H. limbata, Cloeon dipterum, and Ephemera danica, and describe the genomic organization of the Hox gene cluster in E. danica. RESULTS: Transcriptomic sequencing of early instar H. limbata nymphs yielded a high-quality assembly of 83,795 contigs, of which 22,975 were annotated against Folsomia candida, Nilaparvata lugens, Zootermopsis nevadensis and UniRef90 protein databases. Homeodomain protein phylogeny and peptide annotations identified coding sequences for eight of the ten canonical Hox genes (excluding zerknüllt/Hox3 and fushi tarazu) in H. limbata and C. dipterum, and all ten in E. danica. Mayfly Hox protein sequences and embryonic expression patterns of Antp, Ubx, and Abd-A appear highly conserved with those seen in other non-holometabolan insects. Similarly, the genomic organization of the Hox cluster in E. danica resembles that seen in most insects. CONCLUSIONS: We present evidence that mayfly Hox peptide sequences and the embryonic expression patterns for Antp, Ubx, and Abd-A are extensively conserved with other insects, as is organization of the mayfly Hox gene cluster. The protein data suggest mayfly Antp, Ubx, and Abd-A play appendage promoting and repressing roles during embryogenesis in the thorax and abdomen, respectively, as in other insects. The identified expression of eight Hox genes, including Ubx and abd-A, in early instar nymphs further indicates a post-embryonic role, possibly in gill development. These data provide a basis for H. limbata as a complementary Ephemeridae model to the growing repertoire of mayfly model species and molecular techniques.

A new species of Sparsorythus Sroka amp; Soldán, 2008 (Ephemeroptera: Tricorythidae) from Eastern Ghats of Southern India.

A new mayfly species, Sparsorythus sivaramakrishnani sp. nov. from the stream of Pullian cholai, Thuraiyur, Namakkal district, Tamil Nadu, India is described based on male and female nymphs. Sparsorythus sivaramakrishnani sp. nov. differs from the closely related S. gracilis by the shape of the left prostheca, the number of bristle-like processes at base of left prostheca, the ratio and shape of right prostheca, the ratio of femur length: width, the size of the female nymph, the mesonotum overlapping in fifth abdominal segment of the female, and by the shape of hypopharyngeal lingua.