Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

Redescription of adults and first description of the larva of Haemaphysalis (Rhipistoma) adleri Feldman-Muhsam, 1951 (Acari: Ixodidae), parasite of carnivorans in Western Asia.

The adults of Haemaphysalis (Rhipistoma) adleri Feldman-Muhsam, 1951 (Acari: Ixodidae) are redescribed and the larva of this species is described for the first time here. The adults of H. adleri that we studied were collected from various canid, felid and hyaenid carnivorans (Carnivora: Canidae, Felidae, Hyaenidae) as well as a hedgehog (Erinaceomorpha: Erinaceidae) in Iraq, Israel and West Bank. The males, females and larvae of H. adleri can be differentiated from Haemaphysalis (Rhipistoma) species occurring in the Palearctic portion of West Asia and Egypt as well those in the H. asiatica subgroup by the length of idiosomal setae, development and size of spurs on palpi, dental formula on the hypostome and size of spur on coxae. A lectotype of H. adleri has been designated and the geographic distribution and hosts of this tick species are discussed.

Description of the full-grown larva and barcode of Athripsodes taounate taounate Dakki & Malicky 1980 (Trichoptera: Leptoceridae), an Iberic-Maghrebian endemic.

In this paper we describe the main morphological characteristics that distinguish the full-grown larva of Athripsodes taounate taounate, an Iberic-Maghrebian endemic. The conspecificity of the larva and adult was confirmed by DNA analysis. Morphological features that discriminate it from the described Iberian-Maghrebian species of Athripsodes are given.

Morphometric analysis for determination of larval instars in Dermestes frischii Kugelann and Dermestes undulatus Brahm (Coleoptera: Dermestidae).

Dermestes frischii Kugelann, 1792 and Dermestes undulatus Brahm, 1790 are the most abundant species worldwide at outdoor or indoor crime scenes during the dry and skeletal stages of decomposition. The attribution of larval age in these beetles is problematic due to the variable number of instars, which is influenced by environmental factors. In this study, a morphometric approach was used to look for potential morphological features as evidence of larval stages. Breeding and monitoring were performed for both species in an incubator with a preset temperature of 28°C ± 0.5 without a photoperiod. Morphometric measurements were made on 10 larvae per instar for each species using length, width, and thickness parameters. Linear discriminant analysis was then used to generate decision boundaries that clearly separated larval stages. The cross-validation procedure demonstrated that the morphometric approach successfully discriminated adjacent larval stages in both species with high values of sensitivity and specificity. This less-invasive approach could improve the ability to estimate minPMI in forensic studies of Dermestidae beetles. Future studies may extend this approach to other species and establish good practices for collecting and storing specimens for morphometric analysis.

Morphological changes of larvae and pupae of Lucilia sericata (Diptera: Calliphoridae) reared at two temperatures and on three food types.

Determining the minimum postmortem interval (minPMI) from an entomological perspective relies mainly on development data recorded for various species of flies collected from a crime scene or suspicious death. This study focused on the larval and pupal development of Lucilia sericata (Meigen), with an emphasis on the changes of the external morphology of the puparium and its pupal content throughout the duration of metamorphosis. Colonies of L. sericata were reared on 3 types of swine tissue (skeletal muscle, liver tissue, and heart tissue) at 2 different temperature regimes; 24 ±â€…1 °C and 30 ±â€…1 °C. The overall developmental time, larval width and length, and inner and outer pupal morphology changes were observed and recorded. The results show that: (i) temperature significantly influenced overall development time, as well as changes in larval width and length, but this effect was not dependent on tissue type; (ii) larval development duration was longest on heart tissue, and shortest on skeletal muscle for both temperatures; and (iii) pupation was longest for larvae reared on skeletal muscle at 24 ±â€…1 °C, and on liver tissue at 30 ±â€…1 °C, while those larvae reared on liver tissue at 24 ±â€…1 °C and heart tissue at 30 ±â€…1 °C had the shortest pupation period. A seven-character checklist plus 4 landmark stages were developed comprising the external morphology of the puparium and pupal content changes of L. sericata. In conclusion, the study provides larval and pupal development timetables, as well as checklists and photo guides for pupal character development that may be useful for future postmortem determinations.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Revision of the Strodei Subgroup of Nyssorhynchus (Diptera: Culicidae), with descriptions of 2 new species.

Nyssorhynchus (Nyssorhynchus) ibiapabaensis (Sant'Ana & Sallum n. sp.) and Ny. (Nys.) untii (Sant'Ana & Sallum n. sp.) are new species of the Arthuri Complex of the Strodei Subgroup. The new species are described and validated using morphological characters of the male, female, and immature stages. The description of the male, female, fourth-instar larva and pupa of Ny. arthuri (Unti, 1941) and Ny. albertoi (Unti, 1941) are provided for the first time. To avoid nomenclature instability, neotypes are designated for both species. All life stages of Ny. strodei (Root, 1926) employing specimens collected in the Agua Limpa District, Juiz de Fora, Minas Gerais, Brazil are redescribed.

Immature Stages, Natural History and Population Biology of Opoptera syme (Hübner, 1821), (Lepidoptera: Nymphalidae: Satyrinae) from Southeastern Brazil.

The present study describes the immature stages of Opoptera syme (Hübner, [1821]) using SEM and Micro-CT to generate 2D and 3D models to study the morphology and chaetotaxy, and present information on the natural history, behavior, and population biology. In laboratory, eggs were laid singly, and the isolated larvae passed through six to seven instars. Host plant in the study site is unknown, but larvae are known to feed on bamboos; in laboratory, larvae accepted the ornamental bamboo Bambusa textilis McClure, 1940. Adults are diurnal and univoltine, flying from December to April (the austral summer). The present study adds information to the biology and natural history of Brassolini, an iconic and still poorly known tribe of Neotropical butterflies.

Differences in three instars of four carrion nitidulids (Coleoptera, Nitidulidae, Nitidula and Omosita) revealed using SEM.

The genera Omosita and Nitidula from the family Nitidulidae, are often reported to be associated with rotten animal carcasses. However, morphological descriptions of their larval stages are limited and are usually only from the third instar larvae, which does not provide enough systematic data. In this study, the overall structure of three instar larvae from the four Nitidulidae species was compared using optical microscopy, and the resolution was not satisfactory. To compensate, a large number of structures and organs were observed by scanning electron microscopy (SEM). Results showed that the number and distribution of chaetotaxy in different parts, including the macrosetae, setae, and microtrichia, have important identification values between the genera, species, and even instars. We also discuss the possible role of microtrichia in the biology of Nitidulidae larvae. Additionally, we described the number and types of sensilla in three sensory organs, and the morphologic parameters of the head capsule and urogomphi as determined by SEM images, are provided. An identification key with application value for storage products and forensic entomology was also compiled.

On the immature stages of some Merodontini hoverflies (Diptera: Syrphidae) from Europe and Africa.

The genera Eumerus and Merodon (Syrphidae: Merodontini) form together the most speciose grouping of hoverflies in the Palaearctic Region. However, little is known about the morphology and biology of their larvae. The few larvae of Eumerus and Merodon that have been uncovered are phytophagous in underground organs of plants (some Eumerus and all Merodon) or saprophagous in a variety of plants' parts (the reminder of Eumerus). In this study, the second larval stage (L2) of Eumerus lyneborgi Ricarte & Hauser, 2020 and both the larva (L2) and puparium of Merodon constans (Rossi, 1794) are described for the first time. Larvae of E. lyneborgi were found in a decaying stem of Cyphostemma juttae (Dinter & Gilg) Desc., 1960 (Vitaceae) in Namibia (Africa), while larvae of M. constans were collected in bulbs of Leucojum vernum Linnaeus, 1753, (Amaryllidaceae) in France (Europe). Morphology of the immature forms was studied by observation and imaging with stereomicroscopy and scanning electron microscopy. The head skeleton of E. lyneborgi larvae was found to be of the filter feeding type, i.e., in accordance with a saprophagous trophic regime, while that of M. constans was typically phytophagous. Variability in certain characters of the M. constans early stages is described and discussed in relation to the adult form and molecular information published in literature. An updated identification key to all known third larval stages/puparia of Merodon is provided.

A complete morphological characterization of all life stages of the phorid fly Megaselia scalaris.

Megaselia scalaris, commonly known as the scuttle fly, is a cosmopolitan species in the family Phoridae. It is an easily cultured fly species that is an emerging model organism in the fields of genetics and developmental biology. Its affinity for carrion and its predictable life cycle makes it useful in the field of forensic science for estimating the post-mortem interval (PMI) of human remains. Cases of human myasis caused by M. scalaris have also been reported in the medical literature. Despite its ubiquitous prevalence and its relevance across multiple fields, its morphology has not been adequately characterized. Here, we report the complete morphological characterization of all lifestages of M. scalaris, ranging from egg to adult. Scanning electron microscopy has enabled us to uncover morphological features and developmental processes that have previously not been reported in the literature. Our data lays the groundwork for future genetic studies: a morphological characterization of the wild type must be performed before mutants displaying different phenotypes can be identified. In this vein, we also report the observation of a acephalic, or 'headless', adult phenotype whose study could yield insights into the process of cephalogenesis. Finally, all morphological features observed have been compiled into an 'atlas' that should be of use to all workers in the field.

Rediscovery and phylogenetic position of the glassfrog "Centrolene"acanthidiocephalum (Ruiz-Carranza and Lynch, 1989) (Anura: Centrolenidae)with the description of its advertisement call and comments on clutches and tadpoles.

The lack of basic natural history and distributional data represents a challenge for the conservation of rare and endemic amphibian species. This is the case of "Centrolene" acanthidiocephalum, a poorly known glassfrog endemic to the Cordillera Oriental of the Colombian Andes. This species was formally described in the late 1980s and no records have been reported over the last quarter century. Therefore, little is known about its distributional boundaries, morphological variation, natural history, and evolutionary relationships. Here we report the finding of a population of "Centrolene" acanthidiocephalum from a locality around its historical range and provide additional information on its geographic distribution, clutch size, and adult morphology. We also present for the first time data on tadpole morphology, as well as a description of its advertisement call and a phylogenetic tree based on mitochondrial genetic data corroborating its generic position within Centrolene.

A multidisciplinary approach reveals a new species of glassfrog from Colombia (Anura: Centrolenidae: Nymphargus).

Anurans of the family Centrolenidae are a highly diverse clade of Neotropical treefrogs. In the last two decades, glassfrogs have become a model system for studies in ecology and evolutionary biology, in part because their taxonomy and phylogenetic relationships are considered relatively well established. However, there are still many gaps in our knowledge, for example, which morphological characters are important for species delimitation. Consequently, several species complexes in Centrolenidae remain unresolved. Using data on external and internal morphology of adult individuals, tadpoles, advertisement call traits and genetic sequences, we describe a new species of glassfrog (Nymphargus pijao sp. nov.) endemic to Colombia that has been previously missasigned to Nymphargus griffithsi. We include in this description data of three phenotypic characters related to pectoral musculature and testis size, which have been traditionally overlooked in studies about the taxonomy and systematics of glassfrogs. In addition, we present details of a low-cost method implemented in the field to rear tadpoles of the new species. This methodology can solve common problems during the management and care of glassfrogs egg masses and tadpoles, and hence, promotes their description for more species and a better knowledge of the anuran biodiversity in Neotropics.

Larval chaetotaxy and morphometry of Megasternini: Cercyon (Paracercyon) Seidlitz, Cryptopleurum Mulsant and Pelosoma Mulsant (Coleoptera: Hydrophilidae).

The chaetotaxy of the head capsule and head appendages of all the larval instars of Cercyon (Paracercyon) analis and the first and third instars of Cryptopleurum minutum and Pelosoma sp. is described and illustrated for the first time. Morphometric characters derived from the head capsule and mouthparts are also included, together with detailed illustrations of all characters. The morphology and chaetotaxy of these larvae are compared to that of other Megasternini with described larvae. The larval morphology of Megasternini has proven to be very similar, however we provided a group of chaetotaxic characters useful to separate genera. Most of the differences were found in the sensilla of the head capsule, maxilla, and labium, both in size and arrangement, while the chaetotaxy of the antennae and mandibles was strongly conserved.

Description of the larva of Hypsilara (Coleoptera: Elmidae).

The mature larva of the Larainae riffle beetle genus Hypsilara Maier & Spangler, 2011 is described here for the first time, based on larvae of Hypsilara autanai Lassová, Ciampor Jr, Ciamporová-Zatovicová, 2014. The larvae were collected with adults in the stream near the tepui Cerro Autana and Cerro Cuao (southwestern Venezuela) and associated together using DNA barcoding. Larvae of Hypsilara strongly resemble those of Phanoceroides, here we present important morphological diagnostic characters. This description complements the descriptions of the Larainae larvae in the Neotropics, as the larva of Hypsilara was the last one of the group missing so far.

Description of the larva of Macrostemum floridum (Navás 1929) (Trichoptera: Hydropsychidae) and its feeding habits in Kaengkrung National Park, southern Thailand.

A morphological description of the last (fifth) instar larva of Macrostemum floridum (Navás 1929), its life cycle, and feeding habits are provided. The study was conducted along the Khlong Yan Stream in Kaengkrung National Park, southern Thailand. Larval identification was achieved by the metamorphotype method of associating immature stages with the adult. A graph of head capsule widths for all instars is provided. Gut content analysis showed that the main food items were diatoms, green algae, and blue-green algae. The larvae construct fixed retreats and feed as collecting filterers.

Anurocampa (Lepidoptera: Notodontidae): two new species, systematics and immature stages.

Notodontidae (Lepidoptera, Noctuoidea) comprise over 4,000 described species distributed worldwide, among which nearly half are restricted to the Neotropics. Morphology of adults and immatures of Notodontidae have been broadly investigated and many larval, pupal, and adult characters were found to be synapomorphies of subfamilies and tribes. Despite this, the current classification of Notodontidae remains unsettled as most recent classification systems are contradictory due to reliance on incomplete global sampling and, many taxa, especially in the Neotropics, are still informally classified as incertae sedis. Anurocampa Herrich-Shäffer was recently treated as an incertae sedis genus, and immature and adult characters may provide further evidence for its systematic position among the Notodontidae. With this goal in mind, the present study describes the immature stages of Anurocampa mingens Herrich-Shäffer from Brazil and describes two new species in the genus from Costa Rica based on morphology and mitochondrial DNA: Anurocampa markhastingsi Chacón and St Laurent sp. nov. and Anurocampa abelardochaconi Chacón and St Laurent sp. nov. and discusses the systematic position of Anurocampa.

The very rare Japanese endemic diving beetle Japanolaccophilus niponensis (Kamiya, 1939), (Coleoptera: Dytiscidae, Laccophilinae): larval morphology and phylogenetic comparison with other known Laccophilini.

The three larval instars of Japanolaccophilus niponensis (Kamiya, 1939) (Coleoptera: Adephaga, Laccophilinae) are described for the first time according to the now genevralized larval descriptive format of Dytiscidae (Coleoptera: Adephaga), which incorporates detailed chaetotaxic and morphometric analyses. A parsimony analysis based on larval characteristics of 14 Laccophilini species in seven genera was conducted using the program TNT. One of the main results is that Japanolaccophilus Satô, 1972, which so far was treated as being related to Neptosternus Sharp, 1882 now stands out as sister to Laccophilus Leach, 1815, and Philodytes J. Balfour-Browne, 1938 with strong support. Additionally, Laccomimus Toledo & Michat, 2015, and Africophilus Guignot, 1948 are resolved as monophyletic and sister to a clade which itself is subdivided into two well supported clades: Neptosternus + Australphilus Watts, 1978, and Japanolaccophilus + (Laccophilus, Philodytes). Philodytes is here newly accepted as junior synonym of Laccophilus.

Morphology of the abdominal segmental glands and spinning behaviour of Stenus larvae (Coleoptera, Staphylinidae).

We focus on the morphology of the "segmental glands" and their openings in third instar Stenus larvae. The location of the openings was similar in both studied species, with paired rosette-like structures present on the head, all thoracic segments and abdominal segments 1-9. No such openings could be found on the antennae, the maxillary palps, the urogomphi, and the legs as suggested in some older publications. We presume that the glands up to abdominal segment 7 are "adhesive" glands. They are compound glandular units consisting of a secretory syncytium with a common reservoir and a canal cell. The common reservoir is connected through a single efferent duct with the opening of the gland. Glands of abdominal segments 8 and 9 show differences in their length, number of reservoirs, the orientation of the efferent canal, the inner structures of the gland openings towards the exterior and the shape and content of the secretion vesicles indicating that they are silk glands for cocoon building. The spinning behaviour has been observed during the building of the hatching and pupation retreats. The larva first attaches to the substrate with its pygopod, secretes silk droplets from silk gland openings and pulls out a silk filament from the tip of its urogomphi. Whereas L1 and L2 instars produce an open single-layered net, L3 build a closed bi-layered cocoon.

Aedes yunnanensis (Diptera: Culicidae): transferred from the subgenus Hulecoeteomyia to Orohylomyia subg. nov., with descriptions of the species and the new subgenus.

Aedes yunnanensis (Gaschen), currently classified in the subgenus Hulecoeteomyia Theobald, is transferred to a new monobasic subgenus, Orohylomyia Somboon & Harbach, subg. nov., based on morphological assessment of adults, male and female genitalia, larvae, and pupae, and phylogenetic analysis. The new subgenus and its type species are described in detail.