Synergistic effects of chlorantraniliprole and camptothecin on physiological impairments, histopathological, biochemical changes, and genes responses in the larvae midgut of Spodoptera frugiperda.

Spodoptera frugiperda is an economically important agricultural pest and poses a serious threat to food security globally. Its management is gravely challenged by its high polyphagous nature, strong migratory ability, and massive fecundity. Chlorantraniliprole (CHL) is widely utilized in controlling S. frugiperda, its intensive application and over-reliance pose adverse health risks, development of resistance, toxicity to beneficial insects, natural enemies, and environmental contamination. To address S. frugiperda resistance to CHL and its inherent challenges, this study explores the synergistic effects of camptothecin (CPT) with CHL in its management. The binary mixed adversely induced the larvae weight and mortality when compared to single-treated. CHL + CPT (1:20 mg/L) had the highest larvae mortality of (73.80 %) with a high antagonistic factor (0.90), while (1:10 mg/L) with (66.10%) mortality exhibited a high synergistic factor (1.43). Further, CHL + CPT (1:10 mg/L) considerably altered the midgut epithelial cell, peritrophic membrane, microvilli, basement membrane, and regenerative cells. For biochemical analysis, CHL + CPT (1:10 mg/L) significantly decreased glutathione-S-transferase (1-chloro-2,4-dinitrobenzene CDNB) and cytochrome P450 (7-ethoxycoumarin O-deethylation) activities in the midgut in a dose and time dependent manner. Based on RNA-Seq analysis, a total of 4,373 differentially expressed genes (DEGs) were identified from the three treatments. CPT vs CK (Control) had 1694 (968 up-, 726 down-regulated), CHL vs CK with 1771 (978 up-, 793 down-regulated), and CHL + CPT vs CK had 908 (394 up-, 514 down-regulated) DEGs. The enrichment analysis disclosed significant pathways such as metabolism of xenobiotics by cytochrome P450, glutathione metabolism, TOLL and IMD (Immune Deficiency) signaling pathway, longevity regulating pathway. This study provides basis to expatiate on the molecular toxicological mechanism of CHL + CPT in management of fall armyworm.

Immature Stages, Description of Female, and Redescription of Male of Apatelodes kotzschi Draudt, 1929 (Lepidoptera: Bombycoidea: Apatelodidae).

The genus Apatelodes Packard, 1864 comprise more than half of the known Apatelodidae species, but most of its species are placed in the genus without precise justification. The result is a heterogeneous group of species, probably forming a polyphyletic arrangement. Despite being relatively large moths and relatively abundant in light traps, only little information on the natural history and morphology of the Apatelodes immature stages has been published, and only one species is fully described including its immature stages. Aiming to increase the knowledge and provide information towards the definition of the identity of this genus, the present study describes the immature stages, provides a redescription of the male, the first description of the female of Apatelodes kotzschi Draudt, 1929, and we compare and discuss the morphological similarities among Apatelodes species. In general, the immatures of Apatelodes exhibits apparently well-conserved morphological characters, including the gross chaetotaxy configuration. Most differences are found in larval coloration patterns (mainly in the last instar larvae), pupa texture, and cremaster morphology. In contrast, imagos wings and genitalia are rich sources of diagnostic characters which can be used to identify species. However, there are still large gaps in the knowledge of the morphological characters and natural history of most species in the genus that hampers a more robust delimitation of the genus Apatelodes.

Larval and adult digestive tract of the carrion beetle Oxelytrum discicolle (Brullé, 1840) (Coleoptera: Silphidae).

Oxelytrum discicolle is a necrophagous beetle occurring in Central and South America, with potential use in forensic entomology for investigations in the context of legal medicine. The present work aimed to describe the morphology of the digestive tract of larvae and adults and contributes to the knowledge about the digestion associated with necrophagy. In the three larval instars, the foregut is short and narrow, the midgut is a dilated tube, elongated with a smooth surface, and the hindgut is narrow and long, with small lateral projections and a dilated terminal region. The gut epithelium in the second and third instar larvae is vacuolated in the mid- and hindgut, with high production of apocrine secretions in the midgut lumen. In adults, the foregut is short, with small spines in the cuticular intima that covers the flattened epithelium. The midgut is dilated, with many short gastric caeca with regenerative cells in the blind portion, which differentiate in digestive columnar cells towards the midgut lumen. The hindgut is long and narrow, with an enlarged distal portion with folded epithelium lined by a thin cuticle. The histochemical tests reveal the absence of protein storage granules in the gut epithelium of larvae and adults. The gut characteristics of the carrion beetle O. discicolle are similar to those of other predatory and phytophagous Coleoptera, which may indicate that ancestry may influence the alimentary canal morphology more than the feeding habits.

A new desiccation-resistant midge from ephemeral rock pools in South Africa, Polypedilum (Pentapedilum) cranstoni sp. nov. (Diptera: Chironomidae).

One newly described species of Chironomidae, Polypedilum (Pentapedilum) cranstoni sp. nov., was discovered in ephemeral rock pools from the Maloti-Drakensberg mountains in South Africa. Desiccation-resistant larvae were obtained from bottom sediments of rock pools that had been dry for the previous several months. After rehydration, ex situ adults emerged and were collected. The morphology and diagnostic characters of the new species (male and female adults) are described here together with an analysis based on their COI gene sequence.

Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

Larvoscopic study on Dictyocaulus sp. in the faeces of beef cattle in northeastern Brazil / Estudo larvoscópico de Dictyocaulus sp. em fezes de bovinos de corte do nordeste do Brasil

Abstract The lungworm Dictyocaulus viviparus has an important role in cattle health and productivity worldwide, since infections can lead to substantial economic losses. Despite its importance, few studies investigating the epidemiological aspects of infection by this parasite have been conducted. The aim of this study was to report the occurrence of lungworm infection in beef cattle herds reared in an area of livestock production in the northeastern region of Brazil. From September 2020 to August 2021, monthly fecal samples (n = 493) were collected from 46 beef cattle. Among all the animals assessed, lungworm larvae were detected in 8.7% (4/46). None of them presented any clinical sign suggestive of infection by lungworm parasites. Twenty larvae were retrieved, with the minimum number (n = 1) detected in October and December, and the maximum number (n = 13) in November. These presented a mean length of 363 μm (± 28.65 μm) and mean width of 19 μm (± 1.03 μm), and were morphologically similar to Dictyocaulus sp.. This study reports the occurrence of this parasite in this livestock production area. Lastly, local veterinarians need to be aware of inclusion of this parasite in the differential diagnosis of other respiratory infections in beef cattle.

Resumo O parasito pulmonar Dictyocaulus viviparus tem um papel importante na saúde e produtividade bovina em todo o mundo, uma vez que infecções podem levar a perdas econômicas. Além de sua importância, poucos estudos que investigam aspectos epidemiológicos das infecções por esse parasito têm sido realizados. Objetivou-se, nesta pesquisa, relatar a ocorrência de infecção por nematódeos pulmonares em rebanhos bovinos de corte, criados em uma importante área de produção pecuária, na região Nordeste do Brasil. De setembro de 2020 a agosto de 2021, foram coletadas, mensalmente, amostras fecais (n = 493) de 46 bovinos de corte. De todos os animais avaliados, larvas de nematódeos pulmonares foram detectadas em 8,7% (4/46). Os animais não apresentaram nenhum sinal clínico sugestivo de infecção por parasitos pulmonares. Vinte larvas foram recuperadas, com o número mínimo (n = 1) detectado em outubro e dezembro, e o número máximo (n = 13) em novembro. Apresentavam comprimento médio de 363 μm (± 28,65 μm), largura média de 19 μm (±1,03 μm) e eram morfologicamente semelhantes a Dictyocaulus sp.. Este estudo relata a ocorrência desse parasito nesta área. Logo, os veterinários locais devem estar atentos à inclusão desse parasito no diagnóstico diferencial de outras infecções respiratórias em bovinos de corte.

Otobothrium muscatense n. sp. (Trypanorhyncha: Otobothriidae) from Rhabdosargus sarba (Forsskål) (Sparidae), with new locality records of larval trypanorhynchs off the Sultanate of Oman.

In the evaluation of the larval trypanorhynch fauna off the Sultanate of Oman, a total of 188 bony fishes belonging to 16 species were studied, which led to the description of a new species with the establishment of new host and locality records. A new otobothriid species, Otobothrium muscatense n. sp., is described from Rhabdosargus sarba (Forsskål). Based on scolex morphology and tentacular armature, the new species closely resembles the small-sized Otobothrium Linton, 1890 species described as Otobothrium cysticum (Mayer, 1842) and O. crenacolle Linton, 1890. However, it differs from them in the basal armature by a more robust shape of the first principal and the triangular shape of the billhooks as well as by the retractor muscle originating in the posterior part of the tentacle sheath, attaching to a thickening of the tentacle sheath wall, with a muscular branch continuing and attaching at front part of the bulb. It differs from the other species of Otobothrium by size, the combination of six principal hooks and a single intercalary hook in the metabasal armature, and the plane of reflection through the tentacle surfaces. New locality records of the tentaculariids Nybelinia indica Chandra, 1986, Heteronybelinia yamagutii (Dollfus, 1960), Tentacularia coryphaenae Bosc, 1797, and Mixonybelinia lepturi Palm, 2004, together with the lacistorhynchids Pseudogrillotia multiminacantha Palm, 2004 and Callitetrarhynchus gracilis (Rudolphi, 1819), and the pseudotobothriid Parotobothrium balli (Southwell, 1929) off the Muscat region are established. Of the reported species, especially P. multiminacantha and M. lepturi occurring in Trichiurus lepturus L. further demonstrate the relatedness between the Indonesian and the northwestern Indian Ocean fauna, increasing the number of joint records between Indonesia and Omani waters to 14 species.

The potential for histone deacetylase (HDAC) inhibitors as cestocidal drugs.

BACKGROUND: Echinococcosis and cysticercosis are neglected tropical diseases caused by cestode parasites (family Taeniidae). Not only there is a small number of approved anthelmintics for the treatment of these cestodiases, but also some of them are not highly effective against larval stages, such that identifying novel drug targets and their associated compounds is critical. Histone deacetylase (HDAC) enzymes are validated drug targets in cancers and other diseases, and have been gaining relevance for developing new potential anti-parasitic treatments in the last years. Here, we present the anthelmintic profile for a panel of recently developed HDAC inhibitors against the model cestode Mesocestoides vogae (syn. M. corti). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic screening was performed on M. vogae by motility measurements and optical microscopic observations. Some HDAC inhibitors showed potent anthelmintic activities; three of them -entinostat, TH65, and TH92- had pronounced anthelmintic effects, reducing parasite viability by ~100% at concentrations of ≤ 20 µM. These compounds were selected for further characterization and showed anthelmintic effects in the micromolar range and in a time- and dose-dependent manner. Moreover, these compounds induced major alterations on the morphology and ultrastructural features of M. vogae. The potencies of these compounds were higher than albendazole and the anthelmintic effects were irreversible. Additionally, we evaluated pairwise drug combinations of these HDAC inhibitors and albendazole. The results suggested a positive interaction in the anthelmintic effect for individual pairs of compounds. Due to the maximum dose approved for entinostat, adjustments in the dose regime and/or combinations with currently-used anthelmintic drugs are needed, and the selectivity of TH65 and TH92 towards parasite targets should be assessed. CONCLUSION, SIGNIFICANCE: The results presented here suggest that HDAC inhibitors represent novel and potent drug candidates against cestodes and pave the way to understanding the roles of HDACs in these parasites.

Anticancer active trifluoromethylated fused triazinones are safe for early-life stages of zebrafish (Danio rerio) and reveal a proapoptotic action.

The main purpose of this investigation was to evaluate the effect of anticancer active compounds (I-VIII) on zebrafish development in order to select the safest molecules. Larval mortality, embryo hatchability and malformations were end-points used to assess the acute toxicity among embryos and larvae from compounds-/pemetrexed-treated and control groups. LC50 and MNLC (maximal non-lethal concentration) were determined. Lipophilicity-dependent structure-toxicity relationships were established. The results clearly indicated that the majority of test molecules are safe for zebrafish individuals and simultaneously are less toxic than an anticancer agent - pemetrexed. The subsequent aim of this study was to elucidate the molecular mechanism of antiproliferative activity of the most selective compounds. Substantially increased activation of caspase-6 and -8 in cancerous cell lines confirmed the proapoptotic action of molecules examined. Considering the safety for zebrafish individuals, the title compounds as inducers of apoptosis are promising drug candidates in the preclinical phase of drug development.

Application of DNA barcoding and morphometric analysis in differentiating cystacanths of Polymorphus species (Acanthocephala: Polymorphidae) from central Alberta, Canada.

Acanthocephalans are multi-host endoparasites, many of which use freshwater amphipods as intermediate hosts for their larval stages (e.g., cystacanths) while adults live in the intestines of vertebrates, including waterfowl. In central Alberta, Canada, several co-occurring species of the acanthocephalan genus Polymorphus use the amphipod Gammarus lacustris Sars, 1863 as an intermediate host. We applied DNA barcoding and morphometric analysis to differentiate cystacanth larvae from G. lacustris sampled from 17 Albertan water bodies. We slide-mounted specimens and measured morphological traits relating to proboscis hooks. We sequenced the standard DNA barcoding region of the mitochondrial cytochrome c oxidase subunit I gene (COI). Morphometric analysis suggested that the acanthocephalans we collected belonged to four morphologically different groups that keyed to Polymorphus contortus (Bremser, 1821) Travassos, 1926; P. marilis Van Cleave, 1939; P. paradoxus Connel et Corner, 1957; and P. strumosoides (Lundström, 1942) Amin, 2013. Our Bayesian tree based on COI sequences generally corroborated the morphological results and supported that the specimens assigned to P. cf. contortus and P. cf. strumosoides belong to two distinct species. In contrast, the Bayesian tree showed that specimens of P. cf. marilis were nested as a cluster within the P. cf. paradoxus clade. Similarly, small pairwise genetic distance (< 2%) between specimens identified as P. cf. contortus and P. cf. strumosoides suggests that they are conspecific. Future studies should use morphology and sequence data from adult acanthocephalans to assess the taxonomic identity of the cystacanth-based Polymorphus taxa. Our study is the first to provide genetic information for the four Polymorphus taxa and emphasizes the importance of applying multiple approaches to differentiate parasite species.

Evaluation of the Toxicity of Sugammadex in Zebrafish Larvae.

BACKGROUND: Sugammadex is a new neuromuscular blockade reversal agent. Recently, it has been used in patients under general anesthesia. However, sugammadex could be toxic to fetuses and pediatric patients under 3 years of age. In this study, we demonstrated the safety of sugammadex in fetuses, using zebrafish larvae. Furthermore, its neurotoxicity was evaluated using neuronal cell lines. METHODS: We used SH-SY5Y cells to determine the viability of neuronal cells treated with sugammadex. Zebrafish larvae were used to determine the teratogenic effects of sugammadex. RESULTS: Sugammadex showed no adverse effects on neuronal cells and zebrafish larvae. The survival rates of neuronal cells were not different in all concentrations. In addition, the heart formation of zebrafish embryos, which were exposed to various concentrations of sugammadex, were not different. CONCLUSION: This study demonstrated the feasibility of using sugammadex during pregnancy. However, further clinical studies will be required to extrapolate these results to humans.

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Cladistic analysis of the genus Bruggmanniella Tavares (Diptera, Cecicomyiidae, Asphondyliini) with evolutionary inferences on the gall inducer-host plant association and description of a new Brazilian species.

In this study, we present a phylogenetic analysis of the genus Bruggmanniella Tavares based on morphological features. Cladistic analyses were conducted using 57 characters from 26 species. All species of Bruggmanniella except for B. byrsonimae were selected as ingroup and the genera Asphondylia Loew, Bruggmannia Tavares, Illiciomyia Tokuda, Parazalepidota Maia, Pseudasphondylia Monzen, Schizomyia Kieffer, and Lopesia Rübsaamen as outgroup. We used characters from larvae, pupae, adults, and galls. The results of this study supported Bruggmanniella as the sister group of Pseudasphondylia. Bruggmanniella actinodaphnes Tokuda and Yukawa and B. cinnamomi Tokuda and Yukawa have been moved to genus Pseudasphondylia (Pseudasphondylia actinodaphnes (Tokuda and Yukawa) comb. nov. and Pseudasphondylia cinnamomi (Tokuda and Yukawa) comb. nov.). The new genus Odontokeros gen. nov. has been erected for the single species Odontokeros brevipes (Lin, Yang & Tokuda) comb. nov. In addition, we described a new Brazilian species, Bruggmanniella miconia Garcia, Lamas and Urso-Guimarães sp. nov. Identification keys to the New World species of Bruggmanniella are presented.

New records of spirurid nematodes (Nematoda, Spirurida, Guyanemidae, Philometridae & Cystidicolidae) from marine fishes off New Caledonia, with redescriptions of two species and erection of Ichthyofilaroides n. gen.

Recent examinations of spirurid nematodes (Spirurida) from deep-sea or coral reef marine fishes off New Caledonia, collected in the years 2006-2009, revealed the presence of the following five species: Ichthyofilaroides novaecaledoniensis (Moravec et Justine, 2009) n. gen., n. comb. (transferred from Ichthyofilaria Yamaguti, 1935) (females) (Guyanemidae) from the deep-sea fish Hoplichthys citrinus (Hoplichthyidae, Scorpaeniformes), Philometra sp. (male fourth-stage larva and mature female) (Philometridae) from Epinephelus maculatus (Serranidae, Perciformes), Ascarophis (Dentiascarophis) adioryx Machida, 1981 (female) (Cystidicolidae) from Sargocentron spiniferum (Holocentridae, Beryciformes), Ascarophis (Ascarophis) nasonis Machida, 1981 (males and females) from Naso lituratus and N. unicornis (Acanthuridae, Perciformes), and Ascarophisnema tridentatum Moravec et Justine, 2010 (female) from Gymnocranius grandoculis (Lethrinidae, Perciformes). Two species, I. novaecaledoniensis and A. nasonis, are redescribed based on light microscopical (LM) and scanning electron microscopical (SEM) examinations, the latter used in these species for the first time. Morphological data on the specimen of A. tridentatum from the new host species are provided. Philometra sp. (from E. maculatus) most probably represents a new gonad-infecting species of this genus. The newly established genus Ichthyofilaroides n. gen. is characterized mainly by the presence of a small buccal capsule and by the number and distribution of cephalic papillae in the female; it is the sixth genus in the Guyanemidae.

TITLE: Nouvelles mentions de nématodes spirurides (Nematoda, Spirurida, Guyanemidae, Philometridae & Cystidicolidae) de poissons marins de la Nouvelle-Calédonie, avec redescriptions de deux espèces et érection d'Ichthyofilaroides n. gen. ABSTRACT: L'étude récente de nématodes Spirurida de poissons marins de mer profonde ou des récifs coralliens au large de la Nouvelle-Calédonie, collectés dans les années 2006-2009, a révélé la présence des cinq espèces suivantes : Ichthyofilaroides novaecaledoniensis (Moravec et Justine, 2009) n. gen., n. comb. (transféré depuis Ichthyofilaria Yamaguti, 1935) (femelles) (Guyanemidae) du poisson de profondeur Hoplichthys citrinus (Hoplichthyidae, Scorpaeniformes), Philometra sp. (larve mâle de quatrième stade et femelle mûre) (Philometridae) d'Epinephelus maculatus (Serranidae, Perciformes), Ascarophis (Dentiascarophis) adioryx Machida, 1981 (femelle) (Cystidicolidae) de Sargocentron spiniferum (Holocentridae, Beryciformes), Ascarophis (Ascarophis) nasonis Machida, 1981 (mâles et femelles) de Naso lituratus et N. unicornis (Acanthuridae, Perciformes), et Ascarophisnema tridentatum Moravec et Justine, 2010 (femelle) de Gymnocranius grandoculis (Lethrinidae, Perciformes). Deux espèces, I. novaecaledoniensis et A. nasonis, sont redécrites sur la base de la microscopie optique et de la microscopie électronique à balayage, cette dernière étant utilisée pour la première fois chez ces espèces. Des données morphologiques sur le spécimen d'A. tridentatum de la nouvelle espèce hôte sont fournies. Philometra sp. (d'E. maculatus) représente très probablement une nouvelle espèce infectant les gonades de ce genre. Le genre nouvellement établi Ichthyofilaroides n. gen. se caractérise principalement par la présence d'une petite capsule buccale et par le nombre et la répartition des papilles céphaliques chez la femelle. C'est le sixième genre des Guyanemidae.

Contrasting levels of genotype by environment interaction for life history and morphological traits in invasive populations of Zaprionus indianus (Diptera: Drosophilidae).

It has been demonstrated that phenotypic plasticity and genotype by environment interaction are important for coping with new and heterogeneous environments during invasions. Zaprionus indianus Gupta (Diptera: Drosophilidae) is an Afrotropical invasive fly species introduced to the South American continent in 1999. This species is generalist and polyphagous, since it develops and feeds in several different fruit species. These characteristics of Z. indianus suggest that phenotypic plasticity and genotype by environment interaction may be important in this species invasion process. In this sense, our aim was to investigate the role of genetic variation for phenotypic plasticity (genotype by environment interaction) in Z. indianus invasion of the South American continent. Specifically, we quantified quantitative genetic variation and genotype by environment interactions of morphological and life history traits in different developmental environments, that is, host fruits. This was done in different populations in the invasive range of Z. indianus in Argentina. Results showed that Z. indianus populations have considerable amounts of quantitative genetic variation. Also, genotype by environment interactions was detected for the different traits analyzed in response to the different developmental environments. Interestingly, the amounts and patterns of these parameters differed between populations. We interpreted these results as the existence of differences in evolutionary potential between populations that have an important role in the short- and long-term success of the Z. indianus invasion process.

The ontogeny of the lateral line system in Telmatobius atacamensis (Anura, Telmatobiidae).

The lateral line system in anurans is functional during aquatic stages and therefore could provide characters related to larval morphological variation. However, few studies have addressed its components in an integrated overview, and little is known about its ontogenetic variation. This study describes the postembryonic trajectory of the lateral system in Telmatobius atacamensis up to its metamorphic regression. This includes structure, number, topography, and innervation of neuromasts, to contribute new and complete information about its larval organization and its temporal sequence of regression. The arrangement and innervation of lateral lines in T. atacamensis resembles those described for other Type IV tadpoles. Its distinctive features are the orientation of the neuromast stitches in the lateral lines, the presence of supraotic neuromasts, and the first-described case of asymmetry of the ventral trunk line. The temporal sequence of regression during metamorphosis differs between the lateral lines and the lateral line nerves, which remain myelinated into postmetamorphic stages. This asynchronous pattern between different components of the system has also been described for Pseudis paradoxa, which shares with T. atacamensis a remarkably long larval period. This long larval period and gradual metamorphosis could also be related to the constitutive metamorphic regression of the system, in spite of the aquatic lifestyle of these frogs.

New approaches to studying morphological details of intramolluscan stages of Angiostrongylus vasorum / Novas abordagens para estudo morfológico dos estágios intramoluscos de Angiostrongylus vasorum

Abstract Angiostrongylus vasorum is a pulmonary artery parasite of domestic and wild canid. On molluscs, intermediate host, first stage larvae (L1) are found after the first day of infection, in the 8th L2 and in the 30 th L3. It was evaluated L1, L2 and L3 recovered by Baermann technique from Achatina fulica infected with 1000 L1. Fifty larvae/stage were incubated with antibodies anti-β-tubulin, anti-α-tubulin, anti- α-actin, anti-β-actin and anti-collagen, and then with Alexa 633. Fifty larvae/stage were observed with picrosirius red and Oil Red O. It was also observed in the anterior region of L1 the beginning of the chitinous stems development, in the initial portion of the intestine and genital primordium. In L2 anterior region, the papillae, chitinous canes juxtaposed to the mouth and intestines bigger than L1. The L3 musculature is well defined, next to the chitinous stems, there are two round distally arranged from each other. It was observed the whole extension of the intestine genital primordium and intense cellularity in the L3 distal portion. With the picrosirius red the L1, L2 and L3 musculature could be observed, as the nerve ganglia on L3. Oil Red O revealed that L1, L2 and L3 store energy on lipid droplets.

Resumo Angiostrongylus vasorum é um parasito de artérias pulmonares dos canídeos domésticos e silvestres. Nos moluscos, hospedeiros intermediários, encontram-se no primeiro dia após a infecção, larvas de primeiro estágio (L1), ao 8° L2 e ao 30° L3. Avaliou-se L1, L2 e L3 recuperadas pela técnica de Baermann de Achatina fulica infectada com 1.000 L1. Incubou-se 50 larvas/estádio com anticorpos anti-β-tubulina, anti-α-tubulina, anti-β-actina e anti-colágeno e, em seguida, com anticorpo Alexa 633. Observaram-se também 50 larvas/estádio com picrosirius red e Oil Red O, na região anterior da L1, o início do desenvolvimento de hastes quitinosas, a porção inicial do intestino e o primórdio genital. Na região anterior de L2, papilas, bastões quitinosos justapostos à boca e ao intestino maior que em L1. A musculatura de L3 é bem desenvolvida, próximo às hastes quitinosas, há duas estruturas redondas dispostas distalmente uma da outra. Observaram-se também toda a extensão do intestino, o primórdio genital e a intensa celularidade na porção distal da L3. Com o picrosirius red observou-se a musculatura de L1, L2 e L3, assim como, gânglios nervosos na L3. Oil Red O revelou que L1, L2 e L3 armazenam energia em gotículas lipídicas.

A newly discovered radiation of endoparasitic gastropods and their coevolution with asteroid hosts in Antarctica.

BACKGROUND: Marine invertebrates are abundant and diverse on the continental shelf in Antarctica, but little is known about their parasitic counterparts. Endoparasites are especially understudied because they often possess highly modified body plans that pose problems for their identification. Asterophila, a genus of endoparasitic gastropod in the family Eulimidae, forms cysts in the arms and central discs of asteroid sea stars. There are currently four known species in this genus, one of which has been described from the Antarctic Peninsula (A. perknasteri). This study employs molecular and morphological data to investigate the diversity of Asterophila in Antarctica and explore cophylogenetic patterns between host and parasite. RESULTS: A maximum-likelihood phylogeny of Asterophila and subsequent species-delimitation analysis uncovered nine well-supported putative species, eight of which are new to science. Most Asterophila species were found on a single host species, but four species were found on multiple hosts from one or two closely related genera, showing phylogenetic conservatism of host use. Both distance-based and event-based cophylogenetic analyses uncovered a strong signal of coevolution in this system, but most associations were explained by non-cospeciation events. DISCUSSION: The prevalence of duplication and host-switching events in Asterophila and its asteroid hosts suggests that synchronous evolution may be rare even in obligate endoparasitic systems. The apparent restricted distribution of Asterophila from around the Scotia Arc may be an artefact of concentrated sampling in the area and a low obvious prevalence of infection. Given the richness of parasites on a global scale, their role in promoting host diversification, and the threat of their loss through coextinction, future work should continue to investigate parasite diversity and coevolution in vulnerable ecosystems.

Hybrid gene misregulation in multiple developing tissues within a recent adaptive radiation of Cyprinodon pupfishes.

Genetic incompatibilities constitute the final stages of reproductive isolation and speciation, but little is known about incompatibilities that occur within recent adaptive radiations among closely related diverging populations. Crossing divergent species to form hybrids can break up coadapted variation, resulting in genetic incompatibilities within developmental networks shaping divergent adaptive traits. We crossed two closely related sympatric Cyprinodon pupfish species-a dietary generalist and a specialized molluscivore-and measured expression levels in their F1 hybrids to identify regulatory variation underlying the novel craniofacial morphology found in this recent microendemic adaptive radiation. We extracted mRNA from eight day old whole-larvae tissue and from craniofacial tissues dissected from 17-20 day old larvae to compare gene expression between a total of seven F1 hybrids and 24 individuals from parental species populations. We found 3.9% of genes differentially expressed between generalists and molluscivores in whole-larvae tissues and 0.6% of genes differentially expressed in craniofacial tissue. We found that 2.1% of genes were misregulated in whole-larvae hybrids whereas 19.1% of genes were misregulated in hybrid craniofacial tissues, after correcting for sequencing biases. We also measured allele specific expression across 15,429 heterozygous sites to identify putative compensatory regulatory mechanisms underlying differential expression between generalists and molluscivores. Together, our results highlight the importance of considering misregulation as an early indicator of genetic incompatibilities in the context of rapidly diverging adaptive radiations and suggests that compensatory regulatory divergence drives hybrid gene misregulation in developing tissues that give rise to novel craniofacial traits.

Drywood Pest Termite Cryptotermes brevis (Blattaria: Isoptera: Kalotermitidae): a Detailed Morphological Study of Pseudergates.

The Kalotermitidae Cryptotermes brevis (Walker) presents colonies that lack a true worker caste. They have totipotent worker-like individuals named pseudergates. Few studies have characterized the morphology of immature instars, including pseudergates. In order to identify these instars and characterize the pseudergates, we conducted a comparison between morphometric and morphological variations among immature individuals of C. brevis colonies. Juvenile hormone analog (JHA) was used in the first instar nymphs to induce regressive molts and compare morphological differences between nymphs and pseudergates. Results showed the existence of three larval instars and four nymphal instars. These immatures were morphologically characterized. Individuals classified as third instar larvae presented white body, 10 to 12 antennal articles, absent or small non-pigmented compound eyes, and absence of wing buds. Pseudergates presented pigmented abdomen and sclerotized cuticle, 10 to 12 antennal articles, and absent or small compound eyes, and few specimens had large pigmented compound eyes and absence of wing buds. First instar nymphs had pigmented abdomen and sclerotized cuticle, 10 to 12 antennal articles, both large non-pigmented and pigmented compound eyes, the presence of wing buds. Bioassays using JHA on first instar nymphs resulted in a large percentage of nymph-soldier intercastes. We concluded that abdomen pigmentation and sclerotized cuticle are good characters to differentiate pseudergates from larvae and the absence of wing buds is a good character to differentiate pseudergates from nymphs. Our findings not only contribute to the basic biological and morphological information of this species but also help to identify correctly pseudergates in further studies that involve applied bioassays.