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.

A new species of Languidipes Hubbard (Ephemeroptera, Polymitarcyidae) from Borneo.

The genus Languidipes is currently represented by three species distributed in southeastern Asia, India, and Sri Lanka. Languidipes corporaali is the most widely distributed species, and both, male and female imagos, as well as nymphs, are known. In contrast, the other species, L. taprobanes and L. lithophagus, are only known from nymphs. Here, we describe a new species, Languidipes janae sp nov, based on male imagos collected from Borneo, Indonesia. This new species is characterized by the presence of ommation on mesonotum, and penis almost completely divided, with sub-quadrate base and a small outer projection basal to the long and slender distal arms. This constitutes the first record of the genus for Borneo. A cladistic analysis of the subfamily Asthenopodinae supports its taxonomic status.

A new species of Megaglena Peters amp; Edmunds, 1970 (Ephemeroptera: Leptophlebiidae) from Tamil Nadu, India.

Megaglena sivarubani sp. nov. is described based on the nymphs from the Puliyuthu falls of Tamil Nadu, Southern India. The number of species of Megaglena Peters Edmunds is now expanded to three and it is the first record of the genus from the Tamil Nadu part of Southern Western Ghats. Megaglena sivarubani sp. nov. can be distinguished from Indian species M. agasthiya Vasanth, Subramanian Selvakumar by the body size of the nymphs, shape of the labial palp segment III, and by the setation of foretibia. The morphological variations between all the known species of Megaglena are discussed and summarized.

Baetis majus sp. nov., new species of mayfly (Ephemeroptera: Baetidae) from Far East of Russia.

A new species, Baetis majus Tiunova sp. nov., is described and illustrated based on larvae and reared adults discovered in the Russian Far East. The differential identification of this species was determined by the characteristics of other representatives of the genus Baetis Leach, including subgenera Baetis Leach and Tenuibaetis Kang Yang from Eastern and Western Palaearctic, Nearctic and Oriental regions. In addition to morphological studies, DNA barcoding of the described species with average intraspecific K2P distances to nearest neighbours is documented. We reconstructed the phylogenetic relationships of all available cytochrome c oxidase subunit I (COI) sequences of the subgenera of Baetis and Tenuibaetis from four regions. Bayesian analysis using 47 morphological characters additional to partial COI sequences did not allow to determine the species-group of the Baetis genus to which the described species belongs.

A new species of Dudgeodes Sartori, 2008 (Ephemeroptera: Teloganodidae) from Megamalai hills of southern Western Ghats, India.

Dudgeodes sartorii sp. nov. is described based on nymphs from the Megamalai hills of Western Ghats, Southern India. The nymph of Dudgeodes sartorii sp. nov. can be distinguished from other Indian species of Dudgeodes by i) prothorax with two rounded tubercles; (ii) outer margin of mesothorax with simple setae; (iii) distinct maculae in the distal region of femora; (iv) transverse row of setae on fore femora narrower with blunt apex and (v) dark brownish band on the 5th segment of cerci.

A new species of Ephoron Williamson, 1802 (Ephemeroptera: Polymitarcyidae) from Thailand.

The genus Ephoron Williamson, 1802 is widely distributed around the world. In Thailand, only Ephoron indicus Pictet, 1843 was reported in 1961. In this study, a new Ephoron mayfly is described as Ephoron ookaewae sp. nov. In this new species description, the morphological characteristics of larvae and eggs in addition to adults are also shown in detail. Number of tubercles on mandibular tusks, and a median frontal process in larvae, distinguish Ephoron ookaewae sp. nov. from other Ephoron species. Their polar cap shape and a concave indentation in their eggs are also unusual. In addition, the mitochondrial DNA COI sequence data of the newly described Ephoron ookaewae sp. nov. is registered in GenBank. Registration of sequence data for the DNA barcode region of Ephoron mayflies inhabiting the Oriental region remains limited, however it will be useful for future research.

A new species of Caenis Stephens, 1835 (Ephemeroptera: Caenidae) from Tamil Nadu, Southern India.

A new mayfly species Caenis maduraiensis sp. nov. is described and illustrated from nymph and adults collected from lentic habitat around Madurai. The nymph can be distinguished by the following characters: anterior margin of labrum with row of bipectinate setae longer laterally and a group of highly fringed setae behind emargination; and hind claw long, slender and hooked with row of denticles. Adults are characterized by prosternal triangle with slightly concave sides, tip acutely pointed; apical part of forceps abruptly narrowed to the tip, with a long apical spine, and few trichomes on surface.

Description of a new species, nymphs of two known species and a new record of Miroculis/ Edmunds, 1963 (Ephemeroptera: Leptophlebiidae) from Brazil.

Miroculis is one of the most diversified Neotropical genera of Leptophlebiidae in South America with 26 species currently known. In this work we describe a new species of Miroculis based on imagos from Amazonas, and the unknown nymphs of Miroculis (Miroculis) caparaoensis and Miroculis (Ommaethus) misionensis, both associated with imagos. In addition, Miroculis (Ommaethus) misionensis is recorded for the first time from Brazil.

More about Thraulodes Ulmer, 1920 (Ephemeroptera: Leptophlebiidae) from the Brazilian Amazonia: three new species, a new record and a gynandromorph report.

Three new species of Thraulodes Ulmer (Ephemeroptera: Leptophlebiidae) from Brazilian Amazonia are described: Thraulodes yara n. sp., Thraulodes yaciara n. sp. and Thraulodes ykamiaba n. sp., all based on imagoes. All three new species can be easily recognized by general color pattern and genitalia morphology. Thraulodes alboniger Kluge, 2020, known only from Peru, is recorded in Brazil. A remarkable gynandromorph case is presented for T. yara n. sp.

Unmasking a gap: A new oligoneuriid fossil (Ephemeroptera: Insecta) from the Crato Formation (upper Aptian), Araripe Basin, NE Brazil, with comments on Colocrus McCafferty.

The Crato Formation (Araripe Basin) preserves one of the most diverse entomofaunas of the Cretaceous. Among the groups of insects, mayflies stand out in abundance, but among them oligoneuriids are especially rare. A newly discovered adult oligoneuriid from this unit is here described as Incogemina nubila gen. et sp. nov. and new subfamily Incogemininae. A phylogenetic analysis recovered the new taxon as the sister group to the species-rich and cosmopolitan Oligoneuriinae. The paratype of Colocrus indivicum, described as an "oligoneuriid" from the same unit, is here reviewed and considered as belonging to the family Hexagenitidae. The biogeographical and taxonomic implications of this discovery and the phylogenetic position of Incogemina nubila are discussed. Incogemina bridge an important morphological gap between the Oligoneuriinae and the extant Chromarcys. Also, it demonstrates that the divergence between Oligoneuriinae and Incogemininae probably occurred in South America.

Cymbalcloeon gen. nov., an incredible new mayfly genus (Ephemeroptera: Baetidae) from Thailand.

The genus Cymbalcloeon gen. nov. (Ephemeroptera: Baetidae) is established for a new species Cymbalcloeon sartorii sp. nov. from Thailand, based on larval stage. This genus is unique among all of the Baetidae by the presence of three pairs of single gills on segments V-VII, ventrally oriented to cover the sterna VI-IX. Cymbalcloeon sartorii gen. nov. et sp. nov. presents unique or rare morphological characters such as a deeply concave margin between the prostheca and mola, without setae; a very large subtriangular process of the left mandible; a maxillary palp segment II with scarce and very long setae; almost completely fused labial palp segments II and III with numerous very long setae; elongate tarsal claw with two rows of teeth; a shagreen surface of the terga and paraproct; and a very reduced body size. The gills of the new genus move very quickly during respiration and present a near-synchronous protraction. Due to the very derived larval morphological character and the unknown imaginal stage, the exact phylogenetic position of the genus remains unclear; it most certainly belongs to the concept of Anteropatellata and is possibly closely related to the genus Baetopus.

Morphology and evolution of the wing bullae in South American Leptophlebiidae (Ephemeroptera).

Insects were the first animals to take to the skies, and have been flying for over 320 million years. The order Ephemeroptera is, or at least is part of, the most early-diverging lineage of extant winged insects. The extant species present a very short adult life span, mainly dedicated to reproduction and dispersal of eggs. Mating and egg-laying behavior depend on flight. Wings are structures to fly and as such face a number of physical and physiological challenges. The convex curvature along the anterior-posterior axis of the wing generates a camber that must be carefully regulated. One of the most interesting ways of wing bending is provided by the bullae, which have been defined as short sections of flexible chitin, where the flexion lines cross veins. Although the bullae have been frequently used as taxonomic characters, there is no study focused on their morphology, although their prevalence on the wings of mayflies strongly suggests a role in flight. In order to identify evolutionary trends of these structures within Ephemeroptera, we constructed a matrix with comparative anatomy data of the bullae from whole mounts of the wings of 300 specimens belonging to 70 species of several mayfly families, as well as scanning microscopy samples of selected specimens. We also surveyed the number of bullae and their distribution in the wings of the different species within the South American Leptophlebiidae clade. We optimized the characters onto the latest published phylogeny for Leptophlebiidae.

Understanding the captivity effect on invertebrate communities transplanted into an experimental stream laboratory.

Little is known about how design and testing methodologies affect the macroinvertebrate communities that are held captive in mesocosms. To address this knowledge gap, we conducted a 32-d test to determine how seeded invertebrate communities changed once removed from the natural stream and introduced to the laboratory. We evaluated larvae survival and adult emergence in controls from 4 subsequent studies, as well as corresponding within-river community changes. The experimental streams maintained about 80% of the invertebrates that originally colonized the introduced substrates. Many macroinvertebrate populations experienced changes in numbers through time, suggesting that these taxa are unlikely to maintain static populations throughout studies. For example, some taxa (Tanytarsini, Simuliidae, Cinygmula sp.) increased in number, grew (Simuliidae), and possibly recruited new individuals (Baetidae) as larvae, while several also completed other life history events (pupation and emergence) during the 30- to 32-d studies. Midges and mayflies dominated emergence, further supporting the idea that conditions are conducive for many taxa to complete their life cycles while held captive in the experimental streams. However, plecopterans were sensitive to temperature changes >2 °C between river and laboratory. Thus, this experimental stream testing approach can support diverse larval macroinvertebrate communities for durations consistent with some chronic criterion development and life cycle assessments (i.e., 30 d). The changes in communities held captive in the experimental streams were mostly consistent with the parallel changes observed from in situ river samples, indicating that mesocosm results are reasonably representative of real river insect communities. Environ Toxicol Chem 2018;37:2820-2834. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Comparison of cleaning methods for delicate insect specimens for scanning electron microscopy.

The objective of the present study was to compare cleaning methods for delicate insect specimens for investigations with scanning electron microscopy (SEM). As typical specimens we used aquatic larvae of mosquitoes, springtails, larvae of mayflies and caterpillars because they are very fragile and large parts of their body consist of soft tissue. Additionally their cuticle is very often covered with dirt, soil particles or other materials. Cleaning with ultrasonic sound, as the most common cleaning method used for SEM, will destroy fragile insects. Therefore we tested different procedures to remove the dirt particles. In a first approach we compared cleaning with Potassium hydroxide (KOH), Proteinase K, and Triton X in aquatic larvae of flies, which were available in numbers and kept under the same conditions. As our results showed that the treatment with KOH gives the best results we treated in a second approach springtails, larvae of mayflies and caterpillars only with KOH. The springtails and caterpillars were largely free of particles after treatment with KOH; however, the larvae of mayflies were still covered with remnants of diatoms and precipitates of calcium carbonate of the algae. KOH dissolves organic impurities, on the other hand silicon dioxide and lime crusts are not solved. With this limitation, treatment with KOH is a simple technique for routine use as cleaning method for fragile insect specimens for SEM.

Hairworm Infection and Seasonal Changes in Paratenic Hosts in a Mountain Stream in Japan.

For parasites with complex life cycles, the ecological traits determining host competence and seasonal changes in infection in natural habitats are often unclear, making it difficult to predict infection dynamics, including disease outbreaks. Hairworms (phylum Nematomorpha) require both aquatic and terrestrial hosts to complete their life cycle. Although hairworm host competencies have been tested in laboratory experiments, knowledge of the paratenic hosts (aquatic insect larvae) in their natural habitats is limited. This study clarified the species of aquatic insect larvae that are primarily infected by hairworms as paratenic hosts over a year in a mountain stream in central Honshu, Japan. The monthly prevalence and mean abundance of hairworm cysts were high in Ephemera japonica larvae (Ephemeridae: Ephemeroptera) throughout the study period (20.0-88.9 and 0.2-36.8%, respectively). These high prevalence and abundance values may be attributable to their filter-feeding behavior as well as their depositional habitat use. The hairworms also infected leptophlebiids (Ephemeroptera; scrapers), the perlid Calineulia sp., the chloroperlid Haploperla japonica (Plecoptera; predators), and chironomids (Diptera; filter-feeders or predators). The abundance of the cysts tended to be high in aquatic insects inhabiting pools rather than riffles, and the seasonality reflects the reproductive season of the hairworms as well as the phenology of their paratenic hosts. Filter-feeding ephemeropterans inhabiting pools were the major paratenic host of the hairworms in our study site, although their universality and effectiveness as the transporter to definitive hosts remain unclear.

The nymph, habitat, and status of Eatonigenia in China (Ephemeroptera: Ephemeridae).

The nymphs of a new species of Eatonigenia have more modified labrum and smaller foreclaws than congeners, and the males have a more reddish-brown colored body, cerci, genitalia and more sclerotized projections of gonopores. These characters indicate a new species which is named E. zhangi sp. nov. The nymphs live in fine sandy/muddy substrates of large rivers.

A new species of Polyplocia Lestage from Malaysia with comments on the genus (Ephemeroptera, Euthyplociidae, Euthyplociinae).

Polyplocia nebulosa sp. nov. is described based on male and female imagos from Malaysia, Sabah State. Nymphs provisionally assigned to this species are also described. This species can be readily separated from P. vitalisi by the color pattern of the wings, with light brown longitudinal veins, narrow dark brown clouds on cross veins and margins of wings tinged with brown. The male genitalia are distinct from those of other species of Polyplocia: penes are broad, T-shaped, with large lateral projecting lobes, which are apically rounded, and with a small dorsolateral spine on each lobe; the styliger plate is short and not projected posteriorly. Eggs of the genus are described for the first time using scanning electron microscopy. Eggs are 265-267 µm in length, 170-186 µm in width, barrel-shaped, without polar caps or other attachment structures, with one visible micropyle and with chorion forming an irregular mesh with raised ridges (mesh between 3.8-8 µm). A key to male adults of Polyplocia is provided and additional records of Polyplocia from Thailand are given.

A new genus and species of Atalophlebiinae (Insecta: Ephemeroptera: Leptophlebiidae) from Palni hills of the southern Western Ghats, India.

A new genus and new species of Atalophlebiinae, Klugephlebia kodai Selvakumar, Subramanian & Sivaramakrishnan, are described based on male and female imagos and larvae from Palni hills of the southern Western Ghats of India. The imago of Klugephlebia gen. n. can be differentiated from all other genera of Atalophlebiinae by having the combination of vein MP forked slightly less than half of the distance from base to margin, MP2 attached at base to vein MP1 by a crossvein, a distinctive shape of the hindwing, dissimilar claws on each leg, and distinctive genitalia structures. The larva can be distinguished from other Atalophlebiinae larvae by having the combination of gills present on abdominal segments 1-7, dorsal and ventral portions of lamellae of gills 1 slender and lanceolate with branched tracheae, dorsal and ventral portions of lamellae of gills 2-7 wider & lanceolate and long & suddenly tapering at apex, fore and mid femora with regular rows of long thin setae on outer margins, denticles on claws progressively larger apically, a distinctive labrum, the maxillary palp short with long setae on third segment, and the third segment of the labial palp with distinct setation. The larvae cling to the underside of boulders in the middle of streams where the boulders lie amidst sand and silt. Updated keys to the genera of south Indian Leptophlebiidae are provided.

A new species of Prosopistoma Latreille, 1833 (Ephemeroptera: Prosopistomatidae) from South India.

A new species of Prosopistoma (Ephemeroptera: Prosopistomatidae) is described on the basis of mature nymphs from Coorg, Karnataka, South India. Prosopistoma coorgum, new species can be readily distinguished from the other members of Prosopistoma by the following combination of characters: inner margin of outer canine near apex with 2-3 short spines, the length ratio of maxillary palp segments (3.3:4.8:1), ventral margin of fore tibia with 6-8 pectinate setae, and gill II leaf-like and cleft.

Leptophlebiidae (Ephemeroptera) from Roraima State, Brazil: description of two new species of Ulmeritoides Domínguez, 1991.

Two new species of Ulmeritoides Traver, 1959 from Northern Brazil are described based on male imagos. Ulmeritoides aurantifrons sp. nov. differs from all other species of the genus by the following combination of characters: mesonotum with a yellow spot in the apical and medial portions of the mesoscutal suture and a yellowish spot between the apex of the medioscutum and the posterior scutal protuberances, scutellum dark brown with base and apex yellow; forewing hyaline with two cross veins basal to bulla; penis lobes with a ventral groove, apically rounded. Ulmeritoides mirr sp. nov. differs from all other species of the genus by the following combination of characters: mesonotum with mesoscutal suture yellow; apex of the posterior scutal protuberance with a yellow transversal line; forewing hyaline, costal and subcostal areas tinged with light brown; penis lobes with a ventral groove, apically rounded.