Fourth-stage Larvae Research Articles

Teething during sleep: Ultrastructural analysis of pharyngeal muscle and cuticular grinder during the molt in Caenorhabditis elegans.

Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.

Comparative study of transcription profiles of the P-glycoprotein transporters of two Haemonchus contortus isolates: Susceptible and resistant to ivermectin

The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and β-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.

Individuals living in an onchocerciasis focus and treated three-monthly with ivermectin develop fewer new onchocercal nodules than individuals treated annually

BackgroundLittle information is available on the effect of ivermectin on the third- and fourth-stage larvae of Onchocerca volvulus. To assess a possible prophylactic effect of ivermectin on this parasite, we compared the effects of different ivermectin regimens on the acquisition of onchocercal nodules.MethodsWe analyzed data from a controlled randomized clinical trial of ivermectin conducted in the Mbam Valley (Cameroon) between 1994 and 1998 in a cohort of onchocerciasis infected individuals. The number of nodules that appeared between the start and the end of the clinical trial was analyzed, using ANOVA and multivariable Poisson regressions, between four treatment arms: 150 µg/kg annually, 800 µg/kg annually, 150 µg/kg 3-monthly, and 800 µg/kg 3-monthly.ResultsThe mean number of nodules that appeared during the trial was reduced by 17.7% in subjects treated 3-monthly compared to those treated annually (regardless of the dose). Poisson regression model, adjusting on subject’s age and weight, initial number of nodules and intensity of O. volvulus infection in his village of residence, confirmed that the incidence of new nodules was reduced in 3-monthly treatment arms compared to annually treatment arms, and that the dosage of ivermectin does not seem to influence this effect. Furthermore, the number of newly acquired nodules was positively associated with the initial number of nodules. Analysis of disappearance of nodules did not show any significant difference between the treatment groups.ConclusionsTo our knowledge, these results suggest for the first time in humans, that ivermectin has a partial prophylactic effect on O. volvulus. Three-monthly treatment seems more effective than annual treatment to prevent the appearance of nodules.

Functional Characterization of an α-Esterase Gene Associated with Malathion Detoxification in Bradysia odoriphaga

Carboxylesterases (CarEs) are a multigene superfamily of metabolic enzymes involved in metabolic detoxification of xenobiotics. In this study, an α-esterase gene (BoαE1) was identified from Bradysia odoriphaga. Phylogenetic analysis classified BoαE1 into the α-esterase clade. Developmental expression analysis indicated that BoαE1 was significantly expressed in the second to fourth larval stages. Tissue-specific expression analysis indicated that BoαE1 was highly expressed in the larval midgut. After exposure to LC30 of malathion, the CarE activity of B. odoriphaga was induced and the transcriptional level of BoαE1 was significantly up-regulated. Silencing of BoαE1 significantly increased the susceptibility of B. odoriphaga larvae to malathion. Inhibition assays in vitro indicated that malathion significantly inhibited BoαE1 activity. GC-MS assay showed that BoαE1 possesses hydrolase activity toward malathion and participates in the detoxification of malathion. These results strongly suggest that BoαE1 plays a crucial role in detoxification of malathion in B. odoriphaga.

The evaluation of the larvicidal activity of the essential oil extracted from Officinalis rosmarinus on morphometric parameters Culex pipiens

This study was accomplished with the intention of developing a new strategy of conflict against the grubs of C pipiens, vectors of parasitical diseases, by bringing a major interest in the use of essential oils of plants as bio-insecticide. The method of job which we adopted, aimed at the valuation of the larvicide activity of the plant material of R officinalis in this species of mosquito collected from the MEKKERA wadi of the city of SIDI BEL ABBES. The results obtained show that the essential oil extracted from R officinalis manifested toxicity towards fourth-stage larvae in Culex pipiens, with a concentration-response relationship. Analysis of the data shows that R. officinalis causes a reduction in several morphometric parameters such as the width of the thorax, the weight and the body volume of the larval stage 4 individuals of C pipiens compared to the controls.

Anisakis and Hysterothylacium species in Mediterranean and North-East Atlantic fishes commonly consumed in Spain: Epidemiological, molecular and morphometric discriminant analysis

The consumption of raw fish parasitized with larval ascaridoid nematodes of the family Anisakidae can cause anisakiasis, provoking gastrointestinal and/or allergic symptomatology. The main causative agents in the Anisakis genus are the sibling species Anisakis simplex sensu stricto (s.s.) and A. pegreffii of the A. simplex sensu lato (s.l.) complex. Larvae of A. simplex (s.l.) are frequently detected in fish commonly consumed in Spain, as are larvae of the genus Hysterothylacium of the family Raphidascarididae, associated with allergic reactions but not considered pathogenic. Reported here are the results of an epidemiological survey of ascaridoid larvae in three commonly consumed fish species in Spain, horse mackerel (Trachurus trachurus) (n = 52), blue whiting (Micromesistius poutassou) (n = 93) and anchovy (Engraulis encrasicolus) (n = 69), caught in the North-Eastern Atlantic, West Mediterranean and Adriatic Sea. The larvae found in the dissected fish were identified in the following order of abundance: A. simplex (s.l.) (n = 2003), Hysterothylacium aduncum (n = 422), H. fabri (n = 180) and A. physeteris (n = 15). Binomial regression analysis showed a correlation between A. simplex (s.l.) and Hysterothylacium larvae abundance and the host geographical location, the North-Eastern Atlantic being the area with the highest parasitation. Fish length and weight and Fulton's condition factor were correlated with A. simplex (s.l.) abundance only in horse mackerel. There was a significant presence of A. simplex (s.l.) and H. aduncum larvae in the musculature of North-Eastern Atlantic blue whiting, the most parasitized part being the anteroventral region, followed equally by the anterodorsal and central sections. The ITS rDNA of larvae of the sibling species A. simplex (s.s.) and A. pegreffii was identified by PCR-RFLP, and a binary logistic regression model was developed to study their morphometric differentiation. Anisakis simplex (s.s.) was detected in the North-Eastern Atlantic and A. pegreffii in all the areas studied. The morphometric analysis discriminated between the two species at the third and fourth larval stages (L3 and L4), the latter obtained by in vitro culture in RPMI-1640 medium. Two discriminant functions were obtained for the L3 and L4 larvae, the ventricle being a key parameter for specific differentiation in both stages, providing taxonomical criteria that could be used besides molecular identification. The present study reveals differences in the parasitation of the studied fish, including the distribution of larvae in the musculature, related to the host species and its geographical origin.

NEW HOST RECORD OF ECHINOCEPHALUS OVERSTREETI DEARDORFF AND KO (1983) (NEMATODA: GNATHOSTOMATIDAE) FROM THE COMMON SEA BREAM PAGRUS PAGRUS (SPARIDAE) OF THE RED SEA IN EGYPT

Several nematode species of family Gnathostomatidae are considered as fish-borne zoonotic parasites, although feral dogs and cats are the commonly identified definitive hosts of these species, humans may accidentally be part of the life cycle of these nematodes by eating undercooked fish infected by the fourth larval stages (L4). In the present study, thirtyfive fish specimens of the common sea bream Pagrus pagrus (Sparidae) were collected from a location along the Red Sea at Hurghada city, Cairo, Egypt. Twenty two fish were infected by the nematode parasite Echinocephalus overstreeti (family: Gnathostomatidae) isolated from the peritoneal cavity of infected fish attached externally to host viscera. Afterisolation and fixation, worms were examined by light and scanning electron microscopes. Morphologically, parasite possessed most of the features characterizing of Gnathostomatidae members. Body long with very narrow lateral alae, simple pseudolabia, 2 well-developed papillae and amphid, a cephalic bulb armed with six transverse rows of claw-shaped spines and a conical pointed tail equipped with anus.

A Cell Fate Switch in the Caenorhabditis elegans Seam Cell Lineage Occurs Through Modulation of the Wnt Asymmetry Pathway in Response to Temperature Increase.

Populations often display consistent developmental phenotypes across individuals despite inevitable biological stochasticity. Nevertheless, developmental robustness has limits, and systems can fail upon change in the environment or the genetic background. We use here the seam cells, a population of epidermal stem cells in Caenorhabditis elegans, to study the influence of temperature change and genetic variation on cell fate. Seam cell development has mostly been studied so far in the laboratory reference strain (N2), grown at 20° temperature. We demonstrate that an increase in culture temperature to 25° introduces variability in the wild-type seam cell lineage, with a proportion of animals showing an increase in seam cell number. We map this increase to lineage-specific symmetrization events of normally asymmetric cell divisions at the fourth larval stage, leading to the retention of seam cell fate in both daughter cells. Using genetics and single-molecule imaging, we demonstrate that this symmetrization occurs via changes in the Wnt asymmetry pathway, leading to aberrant Wnt target activation in anterior cell daughters. We find that intrinsic differences in the Wnt asymmetry pathway already exist between seam cells at 20° and this may sensitize cells toward a cell fate switch at increased temperature. Finally, we demonstrate that wild isolates of C. elegans display variation in seam cell sensitivity to increased culture temperature, although their average seam cell number is comparable at 20°. Our results highlight how temperature can modulate cell fate decisions in an invertebrate model of stem cell patterning.

Aleyrodes lonicerae Walker (Hemiptera: Aleyrodidae)‘nın Rosa sp. Üzerinde Biyolojik Özellikleri ve Ölümü Esas Alan Yaşam Çizelgeleri

Hanımeli beyazsineği, Aleyrodes lonicerae Walker (Hemiptera: Aleyrodidae)‘nın ergin öncesi dönemlerinin gelişme süreleri gül, Rosa sp. (Rosaceae) bitkileri üzerinde çalışılmıştır. Beyazsinek erginleri Adana‘daki Mercurialis annua L. (Euphorbiaceae) bitkilerinden elde edilmiştir. Gül bitkisi üzerinde 20°C sıcaklıkta A. lonicerae‘nın yumurta, birinci, ikinci, üçüncü, dördüncü larva (pupa) dönemlerinin ve toplam gelişmelerinin süreleri sırası ile, dişi bireyler için 8,44, 5,44, 5,50, 4,50, 5,19, 29,07 erkek bireyler için 8,15, 5,46, 5,92, 4,92, 5,38, 29,83 gün olarak saptanmıştır. Gül bitkisi üzerinde 25°C sıcaklıkta A. lonicerae‘nın yumurta, birinci, ikinci, üçüncü, dördüncü larva (pupa) dönemlerinin ve toplam gelişmelerinin süreleri sırası ile, dişi bireyler için 8,00, 1,67, 2,33, 2,67, 9,67, 24,34; erkek bireyler için 7,67, 2,33, 2,33, 3,33, 7,83, 23,49 gün olarak saptanmıştır. Aleyrodes lonicerae‘nın yumurta, 1., 2., 3. ve 4. larva (pupa) dönemlerinin 20 ve 25°C‘lerdeki ölüm oranları (%) sırasıyla 14,70, 13,97, 25,00, 20,58 ve 4,41; 2,89, 24,65, 26,08, 20,28 ve 13,06 olmuştur. Gül bitkilerinde 20 ve 25°C‘lerdeki en yüksek k değerleri sırası ile üçüncü larva dönemi için 0,2553 ve dördüncü larva dönemi için 0,3010 olarak bulunmuştur.

Aleyrodes lonicerae Walker (Hemiptera: Aleyrodidae)‘nın Viola tricolor L. Üzerinde Biyolojik Özellikleri ve Ölümü Esas Alan Yaşam Çizelgesi

Hanımeli beyazsineği, Aleyrodes lonicerae Walker (Aleyrodidae: Hemiptera)‘nın ergin öncesi dönemlerinin gelişme süreleri laboratuar koşullarında menekşe Viola tricolor L. (Violaceae) bitkileri üzerinde çalışılmıştır. Beyazsinek erginleri Adana‘da Mercurialis annua L. (Euphorbiaceae) bitkilerinden elde edilmiştir. Viola tricolor üzerinde A. lonicerae‘nın yumurta, birinci, ikinci, üçüncü, dördüncü larva (pupa) dönemlerinin gelişme süreleri sırası ile, dişi bireyler için 9,17, 6,33, 5,83, 4,00, 5,17 ve erkek bireyler için 10,27, 5,91, 5,55, 4,82, 4,55 gün olarak saptanmıştır. Yumurtadan ergin oluncaya kadar geçen toplam gelişme süresi dişilerde 30,50 gün ve erkeklerde 31,10 gün olmuştur. Aleyrodes lonicerae‘nın yumurta, 1., 2., 3. ve 4. larva (pupa) dönemlerinde meydana gelen ölüm oranları sırasıyla menekşe yaprakları üzerinde %22,45, 18,37, 12,25, 4,08 ve 8,16 olmuştur. Menekşe bitkileri üzerinde belirlenen döneme bağlı yaşam çizelgesinde, en büyük k-değeri 1. larva döneminde (k=0,1174) saptanmıştır. Saptanan k-değeri aynı zamanda ölüm oranının en yüksek olduğu dönemi göstermektedir. Konukçu bitki tercihini saptamak için yapılan çalışmada, A. lonicerae dişilerinin aynı ortamda bulunan gül (2,67/iki yaprak) ve menekşe (15,83/iki yaprak) üzerine bıraktıkları ortalama yumurta sayıları arasındaki fark önemli bulunmuştur. Menekşe bitkileri üzerindeki denemeler 20±2°C sıcaklık ve %40±5 orantılı neme sahip iklim odalarında yürütülmüştür.

De novo transcriptome sequencing and analysis of Anisakis pegreffii (Nematoda: Anisakidae) third-stage and fourth stage larvae.

Anisakis pegreffii is known as one of the causes of a fish-borne zoonosis, anisakidosis. Despite its significant public health and food hygiene impacts, little is known of the pathogenesis, genetic background of this parasite, at least partly due to the lack of genome and transcriptome information. In this study, RNA-seq and de novo assembly were conducted to obtain transcriptome profiles of the A. pegreffii third and fourth larvae. The third stage larvae (APL3) were collected from chub mackerel and the fourth stage larvae (APL4) were obtained by in vitro culture. In total, 47,243 and 43,660 unigenes were expressed in APL3 and APL4 transcriptomes. Of them, 18,753 were known and 28,490 were novel for APL3, while 18,996 were known and 24,664 were novel for APL4. The most abundantly expressed genes in APL3 were mitochondrial enzymes (COI, COII, COIII) and polyubiquitins (UBB, UBIQP_XENLA). Collagen-related genes (col-145, col-34, col-138, Bm1_54705, col-40) were the most abundantly expressed in APL4. Mitochondrial enzyme genes (COIII, COI) were also highly expressed in APL4. Among the transcripts, 614 were up-regulated in APL3, while 1,309 were up-regulated in APL4. Several protease and protein biosynthesis-related genes were highly expressed in APL3, all of which are thought to be crucial for invading host tissues. Collagen synthesis-related genes were highly expressed in APL4, reflecting active biosynthesis of collagens occurs during moulting process of APL4. Of these differentially expressed genes, several genes (SI, nas-13, EF-TSMT, SFXN2, dhs-27) were validated to highly transcribed in APL3, while other genes (col-40, F09E10.7, pept-1, col-34, VIT) in APL4. The biological roles of these genes in vivo will be deciphered when the reference genome sequences are available, together with in vitro experiments.

A new species of Contracaecum (Nematoda: Anisakidae) found parasitizing Nannopterum brasilianus (Suliformes: Phalacrocoracidae) and Hoplias argentinensis (Characiformes: Erythrinidae) in South America: morphological and molecular characterization of larval and adult stages.

Nematode species of the genus Contracaecum Railliet & Henry, 1912 have been reported around the world in many species of fish-eating birds and seals. Here, Contracaecum jorgei n. sp. is morphologically described using light and scanning electron microscopy for adults and fourth-stage larvae (L4) found in the bird Nannopterum brasilianus and third-stage larvae (L3) found in the freshwater fish Hoplias argentinensis, both from the province of Córdoba, Argentina. Additionally, sequences of cytochrome c oxidase subunit II were obtained from these specimens and molecular phylogenetic analysis was used to determine its relationships within the genus. The present species is distinguished from other species by the number and disposition of cephalic papillae; shape and size of the interlabia; length of the spicules; and number and arrangement of papillae in the posterior end of the male. Furthermore, in the molecular analyses, sequences obtained from adult L4 and L3 specimens of C. jorgei n. sp. were similar and grouped, forming an independent lineage, thus confirming it as a distinct species. Thus, morphological characteristics associated with molecular data support the proposal of a new species.

Cathepsin F of Teladorsagia circumcincta is a recently evolved cysteine protease.

Parasitic cysteine proteases are involved in parasite stage transition, invasion of host tissues, nutrient uptake, and immune evasion. The cysteine protease cathepsin F is the most abundant protein produced by fourth-stage larvae (L4) of the nematode Teladorsagia circumcincta, while its transcript is only detectable in L4 and adults. T. circumcincta cathepsin F is a recently evolved cysteine protease that does not fall clearly into either of the cathepsin L or F subfamilies. This protein exhibits characteristics of both cathepsins F and L, and its phylogenetic relationship to its closest homologs is distant, including proteins of closely related nematodes of the same subfamily.

Eustrongylides sp. (Nematoda, Dioctophymatoidea) parasitizing Hoplias malabaricus (Actinopterygii: Erythrinidae) collected from the state of Rio de Janeiro, Brazil.

Hoplias malabaricus (Characiformes, Erythrinidae), trahira, is a neotropical freshwater fish of economic and public health significance. A total of 45 specimens of H. malabaricus commercialized in the municipality of Magé, state of Rio de Janeiro, Brazil, were acquired between April 2016 and April 2018 to investigate the presence of nematode larvae. Twenty of the fish were found parasitized by 347 fourth-stage nematode larvae identified taxonomically as Eustrongylides sp. using morphological, morphometric and molecular data. The parasitic indices were: prevalence 44.44%, mean intensity 17.35, mean abundance 7.71, and range of infection 2-40. Infection sites were musculature, mesentery, abdominal cavity, and serosa of intestine, stomach and liver. This is the first report of Eustrongylides sp. larvae parasitizing H. malabaricus in the state of Rio de Janeiro.

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

A TGF-β type II receptor that associates with developmental transition in Haemonchus contortusin vitro.

BackgroundThe TGF-β signalling pathway plays a key role in regulating dauer formation in the free-living nematode Caenorhabditis elegans, and previous work has shown that TGF-β receptors are involved in parasitic nematodes. Here, we explored the structure and function of a TGF-β type II receptor homologue in the TGF-β signalling pathway in Haemonchus contortus, a highly pathogenic, haematophagous parasitic nematode.Methodology/Principal findingsAmino acid sequence and phylogenetic analyses revealed that the protein, called Hc-TGFBR2 (encoded by the gene Hc-tgfbr2), is a member of TGF-β type II receptor family and contains conserved functional domains, both in the extracellular region containing cysteine residues that form a characteristic feature (CXCX4C) of TGF-β type II receptor and in the intracellular regions containing a serine/threonine kinase domain. The Hc-tgfbr2 gene was transcribed in all key developmental stages of H. contortus, with particularly high levels in the infective third-stage larvae (L3s) and male adults. Immunohistochemical results revealed that Hc-TGFBR2 was expressed in the intestine, ovary and eggs within the uterus of female adults, and also in the testes of male adults of H. contortus. Double-stranded RNA interference (RNAi) in this nematode by soaking induced a marked decrease in transcription of Hc-tgfbr2 and in development from the exsheathed L3 to the fourth-stage larva (L4) in vitro.Conclusions/SignificanceThese results indicate that Hc-TGFBR2 plays an important role in governing developmental processes in H. contortus via the TGF-β signalling pathway, particularly in the transition from the free-living to the parasitic stages.

First Comparative Redescription of Six Types of Larval Stages of Hysterothylacium sp. (Nematoda: Raphidascarididae) From Red Sea Fishes at Hurghada, Egypt

Nematodes of the genus Hysterothylacium (Nematoda: Raphidascarididae) infect marine fishes either with larval and adult stages. Humans can be accidentally infected upon consumption of infected seafood. Inspire of their importance, little is known about their occurrence and systematics in Egypt. During the present study, different Red Sea fishes at Hurghada (Egypt) were examined for parasitic infection. Among others four types of third-stage larvae and two types of fourth-stage larvae of Hysterothylacium sp. were for the first time redescribed from Egypt and compared from each other as well as from otherwise elsewhere previously described forms. Moreover, their comparative morphometry, fish hosts differences as well as their medical importance, had been discussed.

Identification of Fromiamycalin and Halaminol A from Australian Marine Sponge Extracts with Anthelmintic Activity against Haemonchus contortus.

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus — a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC50) = 26.6 ± 0.74 µM) and L4 motility (IC50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.

Airborne toluene exposure causes germline apoptosis and neuronal damage that promotes neurobehavioural changes in Caenorhabditis elegans

Toluene is a highly volatile organic solvent present in gasoline. Exposure mainly occurs by absorption via the pulmonary tract and easily reaches the central nervous system, which causes toxic effects. Toluene toxicity has been described but not well established. The present work aimed to evaluate the effects of airborne exposure to toluene, the in vivo model Caenorhabditis elegans was assessed to determine whether nematode could be used to evaluate the effects of exposure to toluene and the possible mechanisms of toxicity of the solvent. Worms at the first or fourth larval stages were exposed to toluene for 48 or 24 h, respectively, in a laboratory-developed vapor chamber at concentrations of 450, 850, 1250 and 1800 ppm. We observed increases in worm mortality and significant developmental delays that occurred in a concentration-dependent manner. An increased incidence of apoptotic events in treated germline cells was shown, which was consistent with observed reductions in reproductive capacity. In addition, toluene promoted significant behavioural changes affecting swimming movements and radial locomotion, which were associated with changes in the fluorescence intensity and morphology of GABAergic and cholinergic neurons. We conclude that toluene exposure was toxic to C. elegans, with effects produced by the induction of apoptosis and neuronal damage.

Response of the bacterial community of Propylea japonica (Thunberg) to Cry2Ab protein

Propylea japonica is a very important predator in agricultural ecosystems, which could be exposed to Bt protein. In this study, the bacterial community of P. japonica fed with normal food and food containing Cry2Ab protein was characterized for the first time using qPCR and high-throughput sequencing approaches. Results showed no effect of Cry2Ab on P. japonica development and reproduction. The most abundant bacterial phylum was Firmicutes, and the most abundant genus was Staphylococcus. The total bacteria copy number was not significantly different across four larval stages. Bacteria species composition was gathered more closely in feed on sucrose solution (sucrose-fed) than in larvae only fed on pea aphid (aphid-fed), the diversity indices of some operational taxonomic unit (OTU) were significantly different between sucrose-fed and aphid-fed samples. Different instar larval stages of P. japonica fed with sucrose solution containing Cry2Ab Bt protein and found no effect on microbial community composition and total bacteria copy numbers. However, effects on relative abundance of microbes, copy numbers of Corynebacterium 1 and Glutamicibacter arilaitensis were observed significantly lower in Bt-fed first and fourth larval stages. Low and high concentrations of Cry2Ab protein altered the microbial abundance relative to sucrose-fed P. japonica and copy numbers of G. arilaitensis and Staphylococcus xylosus were significantly lower in Bt-fed samples than control sucrose-fed. Our results are the first report showing that feeding on Cry2Ab protein does not alter microbial species composition in P. japonica, but effects gene copy number of some dominant bacteria. Further investigations are needed to assess the effect of copy number change on P. japonica.