Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera)

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.

Invertebrate hydrobiont fauna transformation in the Dombrovskyi pit lake during the period of 2014-2018

The aquatic invertebrate community of Dombrovskyi pit lake was investigated during 2014–2018. 25 species were recorded there. The only permanent component of plankton in the community is rotifer Brachionus plicatilis Müller, 1786; of benthos and neuston is hemipteran Sigara lateralis (Leach, 1817), beetle Hydrobius fuscipes (Linnaeus, 1758), flies Aedes sp., Ochlerotatus lepidonotus (Edwards, 1920), Culicoides salinarius Kieffer, 1914, Ephydra glauca Meigen, 1830 are permanent components of benthos and neuston. The main diversity of invertebrates is concentrated in littoral zone up to 2 m of depth. This is primarily due to the desalination of these areas by surface runoff. Over the last decade the mineralization of the surface water layer has decreased from 120–138 to 25–28 g/l. Combined with the desalination of water, the increase of species diversity of aquatic invertebrates was noticed in spring seasons (from 7 to 17 taxa). The freshwater taxa, which are not tolerant even to low water salinity, appeared in the community in 2018 (7–25 % of the species diversity), among them Hydrometra stagnorum (Linnaeus, 1758), Rhyacophila tristis Pictet, 1834, Coelambus impressopunctatus (Schaller, 1783), Enochrus coarctatus (Gredler, 1863), Hydrophilus caraboides (Linnaeus, 1758). The role of freshwater taxa, which are tolerant to low water salinity (up to 5 g/l), increased from 0–10 % of the species diversity in 2014–2015 to 22–35 % in 2018. Among them Eucyclops serrulatus (Fischer, 1851), Candona sp., Cypris pubera O. F. Müller, 1776, Cloeon dipterum (Linnaeus, 1760), Libellula depressa Linnaeus, 1758, Sympecta fusca Vander Linden, 1820, Paracorixa concinna (Fieber, 1848) was noted in 2018 for the first time. On the contrary, the number of saltwater taxa decreased from 20–29 % (2014–2015) to 5–14 % (2018). The saltwater aquatic invertebrate community of this pit lake has transformed into brakish-freshwater one over time. 83 % taxa of aquatic invertebrate communities from freshwater puddles near reservoir coast are noted in the Dombrovska reservoir. The introduction of species from protective canals of the reservoir is unlikely, as only 5 % of taxa from there are marked in it. The main way of the forming communities of invertebrates in this reservoir is the periodical flooding of freshwater coastal puddles.

The mayfly subimago explained. The regulation of metamorphosis in Ephemeroptera

ABSTRACTIn the Paleozoic era, more than 400 million years ago, insects continued molting after forming functional wings. Today, however, all flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Ephemeroptera), which molt in the subimago, a flying intermediate stage between the nymph and the adult. However, the identity and homology of the subimago remains underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why do mayflies maintain the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors but nonetheless, clear answers have not yet been found. However, by combining morphological studies, hormonal treatments, and molecular analysis in the mayfly species Cloeon dipterum, we found new answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. Consequently, the subimago is not formed in these treated mayflies. This indicates that metamorphosis is determined prior to the formation of the subimago, which must therefore be considered an instar of the adult stage. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth is spread over the last two stages, which could explain, at least in part, the adaptive sense of the subimago.

Oxygen limitation fails to explain upper chronic thermal limits and the temperature size rule in mayflies

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.

Mayfly response to different stress types in small and mid-sized lowland rivers

Freshwater ecosystems are endangered worldwide by various human pressures, resulting in dramatic habitat and species loss. Many aquatic invertebrates respond to disturbances in their habitat, and mayflies are among the most sensitive ones. Therefore, we investigated mayfly response to anthropogenic disturbances at 46 study sites encompassing slightly to heavily modified small and mid-sized lowland streams and rivers. Mayfly nymphs were sampled between April and September 2016 using a benthos hand net. A total of 21 species was recorded, with Cloeon dipterum (Linnaeus, 1761) being the most frequently recorded one. Nevertheless, the taxa richness was rather low per site, i.e., between zero and nine. Assemblage structure had a high share of lower reaches and lentic (potamic and littoral) elements, and detritivores (gatherers/collectors and active filter feeders). This indicates that hydromorphological alterations lead to assemblage “potamisation” in small and mid-sized rivers. More mayfly species were related to higher oxygen concentration and lower water temperature, abundance of aquatic vegetation and total organic carbon. Additionally, the assemblage diversity and abundance were negatively associated with increasing intensive agriculture area at the catchment scale. This study confirms mayfly bio-indicative properties, i.e., their sensitivity to alterations of their habitat and pollution, but also provides new data related to mayfly response to the impacted environment. Those data can be used for management and protection activities of lowland rivers and their biota according to the requirements of the European Water Framework Directive.

Mouthpart morphology and food habits of a Pampean population of Cloeon dipterum (Linnaeus, 1761) (Ephemeroptera: Baetidae)

Knowledge of the feeding habits of aquatic insects and assignation to different functional feeding groups contributes to a better comprehension of aquatic ecosystems. The feeding habits of larval stages (4–6 mm) of Cloeon dipterum (Linnaeus, 1761) were studied through mouthpart morphology, gut content and were tested in food particle size preference experiments. The description of the mouthparts consisted in the dissection of them and their observation in an optical microscope. Gut content analysis was carried out by ventral dissection of the thorax to isolate the digestive tract. The content of each larva was homogenized, mounted on slides and observed under an optical microscope at 400× magnification with a graduated eyepiece. Food preference experiments consisted on offering fine particulate organic matter (FPOM) and coarse (CPOM) leaves of Laurus nobilis simultaneously. Mouthparts are characterized by robust mandibles with well-developed and asymmetric molar surfaces and maxillae and labium with developed palps, with short setae. Gut content of C. dipterum was dominated by fine detritus represented by 76.9% (SD = 25.7) of the covered area. Also, in the food preference experiments was detected that FPOM consumption was greater than CPOM. Consequently, we consider that the larval stages of C. dipterum are functionally classified as collectors-gatherers preferring fine particle size, and secondary scrapers for CPOM manipulation.

Genomic adaptations to aquatic and aerial life in mayflies and the origin of wings in insects

The first winged insects underwent profound morphological and functional transformations leading to the most successful animal radiations in the history of earth. Despite this, we still have a very incomplete picture of the changes in their genomes that underlay this radiation. Mayflies (Ephemeroptera) are one of the extant sister groups of all other winged insects and therefore are at a key phylogenetic position to understand this radiation. Here, we describe the genome of the cosmopolitan mayfly Cloeon dipterum and study its expression along development and in specific organs. We discover an expansion of odorant-binding proteins, some expressed specifically in the breathing gills of aquatic nymphs, suggesting a novel sensory role for gills. In contrast, as flying adults, mayflies make use of an enlarged set of opsins and utilise these visual genes in a sexually dimorphic manner, with some opsins expressed only in males. Finally, to illuminate the origin of wings, we identify a core set of deeply conserved wing-specific genes at the root of the pterygote insects. Globally, this is the first comprehensive study of the structure and expression of the genome of a paleopteran insect and shows how its genome has kept a record of its functional adaptations.