Identification of fourth-instar larvae of Aedes albopictus (Skuse) (Diptera: Culicidae) employing scanning electron microscopic tool.

Aedes albopictus is a vector of numerous viruses in tropical and subtropical regions worldwide, including encephalitis and dengue fever. Although the identification keys depend exclusively on the characteristics of the fourth larval instar, the studies were carried out using scanning electron microscopy (SEM) on the morphology of Ae. albopictus are still insufficient, and more research is required. Therefore, the purpose of this study was to present a comprehensive description of Ae. albopictus mosquitoes based on the morphological features of their fourth-instar larvae to distinguish them from other mosquito species. Antennae, feeding mouth brushes, mandibles, maxillae, labia, comb scales, anal papillae, siphon, respiratory spiracles, and pecten teeth were among the morphological features specifically described using SEM. The sensilla trichoidea and the sensilla basiconica are the sensory spicules found on the antennal integument. The labrum is a tessellated membrane with two movable brushes on the sides and one middle brush. The mandibles have four sellar setae on the anterolateral side, and the maxillae, which have a thumb-like structure and a palp located laterally and about four-fifths of the way from the maxillary brush base to the maxillary notch, are the articulating appendages. On the apex of the palp, there are five sensilla. The eighth abdominal segment is a tiny pentagonal structure with 12 comb scales laterally and a respiratory siphon dorsally. The siphon has two rows of pectin teeth, with 12 teeth per row. Four pairs of siphonal tufts and five perispiracular lobes surround the respiratory spiracles. The anal segment has four anal papillae, a ventral brush, a dorsal brush, and an incomplete saddle. The findings demonstrate that the fourth-instar larvae of Ae. albopictus have distinctive morphological features, which are crucial for understanding their key characteristics and identifying them, preventing inaccurate identification, and facilitating the control of diseases transmitted by the mosquito vector. RESEARCH HIGHLIGHTS: A detailed description of various taxonomical interest structures for the fourth-instar larvae of Aedes albopictus is included in this study. Scanning electron microscopy is considered a helpful technique for identifying Ae. albopictus larvae in their fourth instar.

Ultrastructure of the dorsal ocellus of Bittacus planus larvae (Mecoptera: Bittacidae) with evolutionary significance.

The Bittacidae are unique in holometabolous insects in that their larvae bear a dorsal ocellus on the frons. The fine structure of the dorsal ocellus, however, has not been investigated to date. Here, the ultrastructure of the larval dorsal ocellus was studied in the hangingfly Bittacus planus Cheng, 1949 using light, scanning, and transmission electron microscopy. The dorsal ocellus of the larvae comprises a cornea, corneagenous cells, and retinula cells. The cornea is a laminated structure. A layer of corneagenous cells is located below the cornea. Numerous retinula cells are arranged tightly beneath the corneagenous cells. The retinula cells modify their adjacent membranes into numerous linear microvilli, which form an analogue of the rhabdom among adjacent retinula cells. The results show that the dorsal ocellus of larval Bittacidae is a highly vestigial organ and appears to be degenerating during the postembryonic development. The presence of the vestigial dorsal ocellus is likely to represent an ancestral plesiomorphy of holometabolous insects, providing new evidence for exploring the evolutionary origin of holometabolous larvae.

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space.

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic-related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in-vivo imaging with cryo-electron microscopy and cryo-elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.

Solanum nigrum Extract and Solasonine Affected Hemolymph Metabolites and Ultrastructure of the Fat Body and the Midgut in Galleria mellonella.

Glycoalkaloids, secondary metabolites abundant in plants belonging to the Solanaceae family, may affect the physiology of insect pests. This paper presents original results dealing with the influence of a crude extract obtained from Solanum nigrum unripe berries and its main constituent, solasonine, on the physiology of Galleria mellonella (Lepidoptera) that can be used as an alternative bioinsecticide. G. mellonella IV instar larvae were treated with S. nigrum extract and solasonine at different concentrations. The effects of extract and solasonine were evaluated analyzing changes in carbohydrate and amino acid composition in hemolymph by RP-HPLC and in the ultrastructure of the fat body cells by TEM. Both extract and solasonine changed the level of hemolymph metabolites and the ultrastructure of the fat body and the midgut cells. In particular, the extract increased the erythritol level in the hemolymph compared to control, enlarged the intracellular space in fat body cells, and decreased cytoplasm and lipid droplets electron density. The solasonine, tested with three concentrations, caused the decrease of cytoplasm electron density in both fat body and midgut cells. Obtained results highlighted the disturbance of the midgut and the fat body due to glycoalkaloids and the potential role of hemolymph ingredients in its detoxification. These findings suggest a possible application of glycoalkaloids as a natural insecticide in the pest control of G. mellonella larvae.

Description of larval morphology and phylogenetic relationships of Heterotemna tenuicornis (Silphidae).

Providing clear and detailed morphological descriptions of endemic species in limited areas enables new knowledge of their biology and ecology to be obtained through citizen science. This information can be further used for their protection. Our study presents the first morphological description of the larvae of all three instars of Heterotemna tenuicornis (Brullé, 1836), an endemic species of the Canary Islands that, together with H. britoi García & Pérez, 1996 and H. figurata (Brullé, 1839), belongs to the peculiar genus Heterotemna Wollaston, 1864. Furthermore, we present the first sequences of two mitochondrial genes (COI, 16S) obtained from larval specimens, and cross reference them with sequences from an adult specimen. Phylogenetic analysis of molecular data placed the genus Heterotemna within the genus Silpha Linnaeus, 1758, suggesting paraphyly of Silpha. In our study, we underline the importance of using a combination of morphological description and molecular data, that can be used for barcoding developmental stages which could not otherwise be definitely associated.

High-precision targeting workflow for volume electron microscopy.

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

Echinocephalus inserratus sp. n. (Nematoda: Gnathostomatidae) from the stingray Pastinachus ater (Dasyatidae) and new records of congeneric and some other nematode larvae from teleost fishes off New Caledonia.

Based on light and electron microscopical studies, a new nematode parasite, Echinocephalus inserratus sp. n. (Spirurida: Gnathostomatidae), is described from the spiral valve of the broad cowtail stingray Pastinachus ater (Macleay) (Dasyatidae, Myliobatiformes) from off New Caledonia. The new species is morphologically and biometrically most similar to Echinocephalus overstreeti Deardorff et Ko, 1983, differing from it mainly in the absence of serrations on the posterior parts of pseudolabia and on interlabia, and in having a longer gubernaculum (150-299 µm long). Morphologically unidentifiable, mostly encapsulated larvae of Echinocephalus spp. were recorded from the following six species of teleost fishes collected in New Caledonian waters, serving as paratenic hosts: Perciformes: Acanthopagrus berda (Forsskål) (Sparidae) and Nemipterus furcosus (Valenciennes) (Nemipteridae); Tetraodontiformes: Abalistes stellatus (Anonymous), Pseudobalistes fuscus (Bloch et Schneider) (both Balistidae), Lagocephalus sceleratus (Gmelin) (Tetraodontidae) and Aluterus monoceros (Linnaeus) (Monacanthidae). Co-parasitising larvae of Ascarophis sp. and Hysterothylacium sp. were also collected from P. fuscus. All these findings represent new host and geographical records. A key to valid species of Echinocephalus Molin, 1858 is provided.

Form and function of labral fan-ray microtrichia and posterior proleg barbs in Neotropical black flies larvae (Diptera: Simuliidae).

The microtrichia of the labral fan primary rays, considered as the finest structure of the larval filter mechanism in Simuliidae, and the posterior proleg hooks, a structure used for substrate attachment, were describe using Scanning Electron Microscopy for the first time in 14 Neotropical species of black flies. Four patterns of primary rays microtrichia and two types of hooks barbs disposition were found. The present study provides new morphological information for Neotropical black fly species, compares it with previous information for worldwide species. Also, shows that the presence of posterior proleg hooks barbs is common among black fly species, that these barbs are not related with the larval habitat or with the primary ray microtrichia pattern, and that flow conditions may influence the last-instar larvae microtrichial patterns, at least in most of the Neotropical species studied here.

Description of the larval and adult hindgut tract of the common spider crab Maja brachydactyla Balss, 1922 (Brachyura, Decapoda, Malacostraca).

Arthropods are the most diversified animals on Earth. The morphology of the digestive system has been widely studied in insects; however, crustaceans have received comparatively little attention. This study describes the hindgut tract of the common spider crab Maja brachydactyla Balss, 1922, in larvae and adults using dissection, light and electron microscopical analyses. The hindgut tract maintains a similar general shape in larvae and adults. Major differences among stages are found in the morphology of epithelial cells and microspines, the thickness of the cuticle and connective-like tissue, and the presence of rosette glands (only in adults). Here, we provide the description of the sub-cellular structure of the folds, epithelium (conformed by tendon cells), musculature, and microspines of the hindgut of larvae and adults of M. brachydactyla. The morphological features of the hindgut of M. brachydactyla are compared with those of other arthropods (Insecta, Myriapoda and Arachnida). Our results suggest that the morphology of the hindgut is associated mainly with transport of faeces. In adults, the hindgut may also exert an osmoregulatory function, as described in other arthropods. At difference from holometabolous insets, the hindgut of M. brachydactyla (Decapoda) does not undergo a true metamorphic change during development, but major changes observed between larval and adult stages might respond to the different body size between life stages.

Small compounds mimicking the adhesion molecule L1 improve recovery in a zebrafish demyelination model.

Demyelination leads to a loss of neurons, which results in, among other consequences, a severe reduction in locomotor function, and underlies several diseases in humans including multiple sclerosis and polyneuropathies. Considerable clinical progress has been made in counteracting demyelination. However, there remains a need for novel methods that reduce demyelination while concomitantly achieving remyelination, thus complementing the currently available tools to ameliorate demyelinating diseases. In this study, we used an established zebrafish demyelination model to test selected compounds, following a screening in cell culture experiments and in a mouse model of spinal cord injury that was aimed at identifying beneficial functions of the neural cell adhesion molecule L1. In comparison to mammalian nervous system disease models, the zebrafish allows testing of potentially promotive compounds more easily than what is possible in mammals. We found that our selected compounds tacrine and duloxetine significantly improved remyelination in the peripheral and central nervous system of transgenic zebrafish following pharmacologically induced demyelination. Given that both molecules are known to positively affect functions other than those related to L1 and in other disease contexts, we propose that their combined beneficial function raises hope for the use of these compounds in clinical settings.

Ultrastructural observations on the oncomiracidium epidermis and adult tegument of Discocotyle sagittata, a monogenean gill parasite of salmonids.

During their different life stages, parasites undergo remarkable morphological, physiological, and behavioral "metamorphoses" to meet the needs of their changing habitats. This is even true for ectoparasites, such as the monogeneans, which typically have a free-swimming larval stage (oncomiracidium) that seeks out and attaches to the external surfaces of fish where they mature. Before any obvious changes occur, there are ultrastructural differences in the oncomiracidium's outer surface that prepare it for a parasitic existence. The present findings suggest a distinct variation in timing of the switch from oncomiracidia epidermis to the syncytial structure of the adult tegument and so, to date, there are three such categories within the Monogenea: (1) Nuclei of both ciliated cells and interciliary cytoplasm are shed from the surface layer and the epidermis becomes a syncytial layer during the later stages of embryogenesis; (2) nuclei of both ciliated cells and interciliary syncytium remain distinct and the switch occurs later after the oncomiracidia hatch (as in the present study); and (3) the nuclei remain distinct in the ciliated epidermis but those of the interciliary epidermis are lost during embryonic development. Here we describe how the epidermis of the oncomiracidium of Discocotyle sagittata is differentiated into two regions, a ciliated cell layer and an interciliary, syncytial cytoplasm, both of which are nucleated. The interciliary syncytium extends in-between and underneath the ciliated cells and sometimes covers part of their apical surfaces, possibly the start of their shedding process. The presence of membranous whorls and pyknotic nuclei over the surface are indicative of membrane turnover suggesting that the switch in epidermis morphology is already initiated at this stage. The body tegument and associated putative sensory receptors of subadult and adult D. sagittata are similar to those in other monogeneans.

Tadpole of the Amazonia frog Edalorhina perezi (Anura: Leptodactylidae) with description of oral internal and chondrocranial morphology.

The genus Edalorhina consists of two species of small forest-floor frogs inhabiting the Amazon basin. The tadpole of Edalorhina perezi, the most widely distributed species, was previously described based on a single and early stage (Gosner 25) individual. Herein, we provide a description of the tadpole in Gosner stages 35-36 including internal morphology data (i.e., buccopharyngeal cavity and larval skeleton) based on samples from two populations from Ecuador. Edalorhina shares a generalized morphology with most members of its closely related taxa; however, it is distinguished from the other species by having an almost terminal oral disc. The presence of a dextral vent tube is considered a synapomorphy for the clade consisting of Edalorhina, Engystomops, and Physalaemus. Within this clade, the combination of two lingual papillae, a filiform median ridge, and the lack of buccal roof papillae are diagnostic of E. perezi and putative autapomorphies of Edalorhina. Chondrocranial anatomy provides characteristics, that is, presence of and uniquely shaped processus pseudopterygoideus and cartilago suprarostralis with corpora and alae joined by dorsal and ventral connections that readily differentiates the genus from other Leiuperinae.

Ixodes silvanus n. sp. (Acari: Ixodidae), a new member of the subgenus Trichotoixodes Reznik, 1961 from northwestern Argentina.

Females, nymphs, and larvae of Ixodes silvanus n. sp. collected from birds and from the vegetation in northwestern Argentina (Yungas Phytogeographic Province) are described herein. The new species belongs to the subgenus Trichotoixodes (Acari: Ixodidae). The female is diagnosed by a combination of the following characters: scutum with setae moderately long and more numerous in central field, fewer and moderately long setae on lateral fields, and inconspicuous setae in anterior field; basis capituli subtriangular dorsally; porose areas large and irregular in shape, lacking distinct margins; auriculae with straight edges diverging posterolaterally and ending with small blunt processes; hypostome narrow and pointed with dental formula 4/4 in the anterior third, then 3/3 and 2/2 near the base; coxae I with two spurs, sub-equal in size, internal slightly slimmer than external. The nymph is diagnosed by notum with numerous and long setae, ventral surface covered by numerous whitish setae, scutum with short scapulae and few and shallow punctations, setae on scutum few, short and irregularly distributed, basis capituli sub-triangular dorsally with posterior margin straight, cornua large and directed postero-laterally, auriculae large and projected laterally, lateral margin of basis capituli above auriculae with a lateral and triangular projection, hypostome pointed with dental formula 3/3 in the anterior third and then 2/2, and coxa I with two short, sub-equal, triangular spurs. The diagnostic characters of the larva are: basis capituli dorsally sub-triangular with lateral angles acute and posterior margin straight, auriculae as large triangular lateral projections, hypostome with apex bluntly pointed and dental formula 3/3 in the anterior third and then 2/2, coxa I with two short, sub-equal, triangular spurs, and pattern of dorsal and ventral body setae. This new species is phylogenetically related to Ixodes brunneus, Ixodes turdus and Ixodes frontalis, and the principal hosts for all its parasitic stages are birds.

Postembryonic development and differentiation of the midgut in the freshwater shrimp Neocaridina davidi (Crustacea, Malacostraca, Decapoda) larvae.

Neocaridina davidi is a freshwater shrimp that originates from Taiwan and is commonly bred all over the word. Like all decapods, which develop indirectly, this species has pelagic larvae that may differ entirely in their morphology and habits from adult specimens. To fill a gap of knowledge about the developmental biology of freshwater shrimps we decided to document the 3D-localization of the midgut inside the body cavity of larval stages of N. davidi using X-ray microtomography, and to describe all structural and ultrastructural changes of the midgut epithelium (intestine and hepatopancreas) which occur during postembryonic development of N. davidi using light and transmission electron microscopy. We laid emphasis on stem cell functioning and cell death processes connected with differentiation. Our study revealed that while the intestine in both larval stages of N. davidi has the form of a fully developed organ, which resembles that of adult specimens, the hepatopancreas undergoes elongation and differentiation. E-cells, which are midgut stem cells, due to their proliferation and differentiation are responsible for the above-mentioned processes. Our study revealed that apoptosis is a common process in both larval stages of N. davidi in the intestine and proximal region of the hepatopancreas. In zoea III, autophagy as a survival factor is activated in order to protect cells against their death. However, when there are too many autophagic structures in epithelial cells, necrosis as passive cell death is activated. The presence of all types of cell death in the midgut in the zoea III stage confirms that this part of the digestive tract is fully developed and functional. Here, we present the first description of apoptosis, autophagy and necrosis in the digestive system of larval stages of Malacostraca and present the first description of their hepatopancreas elongation and differentiation due to midgut stem cell functioning.

Morphological description of the third instar larva of Lasiotrichius succinctus hananoi (Sawada) (Coleoptera: Scarabaeidae: Cetoniinae: Trichiini), using scanning electron microscopy.

Cetoniinae is one of the showiest scarab groups, exhibiting bright-metallic body colors, and usually attract great attention from entomologists and amateur collectors. Larvae of Cetoniinae show dramatically diversity on morphology and living habits. Although being considered one of the best-studied groups of Scarabaeidae, larvae have been described for less than 5% species to the known Cetoniinae. In this study, the final instar larva of Lasiotrichius succinctus hananoi was described using scanning electron microscopy. The larvae are peculiar for bearing a haptomeral process dividing 10 spines into two groups: six on the left side, four on the right side, different from the previous descriptions on L. succinctus (Pallas, 1781). The morphological differences under SEM imply the further requirement of taxonomic revision in Lasiotrichius. Both advantage and disadvantage of SEM utilizing in larval descriptions were briefly discussed.

Larvicidal activity, route of interaction and ultrastructural changes in Aedes aegypti exposed to entomopathogenic fungi.

Blastospores or conidia (formulated or not) of entomopathogenic fungi were assessed against Aedes aegypti larvae. Larvae (L2) were exposed to 105, 106, 107, and 108 propagules mL-1 water suspension. Mineral oil at 0.1%, 0.5%, or 1.0% (v/v) was employed to observe the effect on larval survival. The 0.1% mineral oil did not affect larval survival. Accordingly, 107 propagules mL-1 and 0.1% mineral oil were used to prepare all fungal emulsions. The fungal suspension or formulation was prepared as follows: 107 propagules mL-1 on 0.03% TweenⓇ 80 (v/v) aqueous solution or 107 propagules mL-1 on 0.03% TweenⓇ 80 plus 0.1% mineral oil; larval survival rates were evaluated for 7 days, and median survival time (S50) was also determined. The presence of fungi in larvae was examined both histologically and by scanning electron microscopy 24 h or 48 h after exposure. To evaluate the larval growth, larvae were exposed to 107 propagules mL-1 for 48 hours and their length measured using a digital caliper. Here, propagules had similar results in reducing the larvae survival rate and time. The treatment with Beauveria bassiana s.l. at 108 propagules mL-1 or with Metarhizium anisopliae s.l. at 108 blastopores mL-1 reduced the larval survival time to two days. M. anisopliae s.l. at 108 conidia mL-1 reduced the survival time to three days. The survival time of larvae submitted to the other treatments ranged from 6 days to over 7 days. M. anisopliae s.l. or B. bassiana s.l. oil-in-water emulsions at 107 propagules mL-1 yielded better results than the water suspensions, the larvae survival rate was 2 days for both propagules in oil-in-water emulsion. Larvae exposed to blastospores from both isolates or M. anisopliae conidia were longer than in the other treatments. Scanning electron microscopy and histology analyzes found fungi predominantly in the gut, mouthparts, and perispiracular lobes of larvae. Formulated fungus yielded better results than the aqueous suspensions for control of mosquito larvae. Thus, for the first time, the effect of mineral oil on the fungal interaction on A. aegypti larvae was observed as well as the effect of entomopathogenic fungi in the growth of larvae, supporting the search for strategies to control this arthropod.

Anatomical changes in postembryonic development of Pycnogonum litorale.

Sea spiders (Pycnogonida) are a small group of arthropods, sister to other chelicerates. They have an unusual adult bauplan, oligosegmented larvae, and a protracted postembryonic development. Pycnogonum litorale (Strøm, 1762) is an uncommonly long-lived sea spider with a distinctive protonymphon and adult anatomy. Although it was described ~250 years ago, little is known about its internal organization and development. We examined the anamorphic and early epimorphic development of this species using histology, light microscopy, and SEM, and provide the first comprehensive anatomical study of its many instars. Postembryonic development of P. litorale includes transformations typical of pycnogonids: reorganization of the larval organs (digestive, nervous, secretory), formation of the abdomen, trunk segments (+ appendages), primary body cavity and reproductive system. Specific traits include the accelerated articulation of the walking legs, formation of the subesophageal and posterior synganglia, and the system of twin midgut diverticula. In addition, P. litorale simultaneously lose the spinning apparatus and all larval appendages. We found that developmental changes occur in synchrony with changes in ecology and food sources. The transition from the anamorphic to the epimorphic period in particular is marked by considerable anatomical and lifestyle shifts. HIGHLIGHTS: Postembryonic development of P. litorale includes numerous anamorphic and epimorphic stages. The instars acquire abdomen, trunk segments, body cavity, and gonads, while losing all larval appendages. Developmental changes are synchronized with changes in lifestyle and food sources.

Anatomy and histology of the alimentary canal of larvae and adults of Chrysoperla externa (Hagen, 1861) (Neuroptera: Chrysopidae).

The larvae of the lacewing Chrysoperla externa are important predators with the potential to be used in the biological control in agriculture. Although some studies provide important data on the gut morphology in lacewings, they are limited to few species. This study describes the anatomy and histology of the alimentary canal in the predatory larvae and herbivorous adult of C. externa. In larvae, the crop is the larger part of the foregut and it is connected to the midgut by the stomodeal valve. The midgut is an enlarged sac-like organ. At the mid-hindgut transition, there are eight Malpighian tubules. The hindgut is a non-functional vestigial region in the larvae. In adults, the crop has a diverticulum associated with large tracheal trunks, a conic proventriculus with sclerotized lips followed by an elongated tubular midgut. Histological analyses of larval and adult midgut show the presence of a single-layered epithelium with columnar cells with well-developed brush border, nests of regenerative cells, and a peritrophic matrix lining the midgut lumen. The hindgut in adults has an epithelium with cubic cells lined by a thin cuticular intima and rectal pads in the rectum. These data are discussed in comparison with the digestive tract in other Chrysopidae.

Synchrotron microtomography applied to the volumetric analysis of internal structures of Thoropa miliaris tadpoles.

Amphibians are models for studying applied ecological issues such as habitat loss, pollution, disease, and global climate change due to their sensitivity and vulnerability to changes in the environment. Developmental series of amphibians are informative about their biology, and X-ray based 3D reconstruction holds promise for quantifying morphological changes during growth-some with a direct impact on the possibility of an experimental investigation on several of the ecological topics listed above. However, 3D resolution and discrimination of their soft tissues have been difficult with traditional X-ray computed tomography, without time-consuming contrast staining. Tomographic data were initially performed (pre-processing and reconstruction) using the open-source software tool SYRMEP Tomo Project. Data processing and analysis of the reconstructed tomography volumes were conducted using the segmentation semi-automatic settings of the software Avizo Fire 8, which provide information about each investigated tissues, organs or bone elements. Hence, volumetric analyses were carried out to quantify the development of structures in different tadpole developmental stages. Our work shows that synchrotron X-ray microtomography using phase-contrast mode resolves the edges of the internal tissues (as well as overall tadpole morphology), facilitating the segmentation of the investigated tissues. Reconstruction algorithms and segmentation software played an important role in the qualitative and quantitative analysis of each target structure of the Thoropa miliaris tadpole at different stages of development, providing information on volume, shape and length. The use of the synchrotron X-ray microtomography setup of the SYRMEP beamline of Elettra Synchrotron, in phase-contrast mode, allows access to volumetric data for bone formation, eye development, nervous system and notochordal changes during the development (ontogeny) of tadpoles of a cycloramphid frog Thoropa miliaris. As key elements in the normal development of these and any other frog tadpole, the application of such a comparative ontogenetic study, may hold interest to researchers in experimental and environmental disciplines.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.