A double ovulation protocol for Xenopus laevis produces doubled fertilisation yield and moderately transiently elevated corticosterone levels without loss of egg quality.

The African claw-toed frog, Xenopus laevis, is a well-established laboratory model for the biology of vertebrate oogenesis, fertilisation, and development at embryonic, larval, and metamorphic stages. For ovulation, X. laevis females are usually injected with chorionic gonadotropin, whereupon they lay typically hundreds to thousands of eggs in a day. After being rested for a minimum of three months, animals are re-used. The literature suggests that adult females can lay much larger numbers of eggs in a short period. Here, we compared the standard "single ovulation" protocol with a "double ovulation" protocol, in which females were ovulated, then re-ovulated after seven days and then rested for three months before re-use. We quantified egg number, fertilisation rate (development to cleavage stage), and corticosterone secretion rate as a measure of stress response for the two protocol groups over seven 3-month cycles. We found no differences in egg number-per-ovulation or egg quality between the groups and no long-term changes in any measures over the 21-month trial period. Corticosterone secretion was elevated by ovulation, similarly for the single ovulation as for the first ovulation in the double-ovulation protocol, but more highly for the second ovulation (to a level comparable to that seen following shipment) in the latter. However, both groups exhibited the same baseline secretion rates by the time of the subsequent cycle. Double ovulation is thus transiently more stressful/demanding than single ovulation but within the levels routinely experienced by laboratory X. laevis. Noting that "stress hormone" corticosterone/cortisol secretion is linked to physiological processes, such as ovulation, that are not necessarily harmful to the individual, we suggest that the benefits of a doubling in egg yield-per-cycle per animal without loss of egg quality or signs of acute or long-term harm may outweigh the relatively modest and transient corticosterone elevation we observed. The double ovulation protocol therefore represents a potential new standard practice for promoting the "3Rs" (animal use reduction, refinement and replacement) mission for Xenopus research.

The effect of repeat feeding on dengue virus transmission potential in Wolbachia-infected Aedes aegypti following extended egg quiescence.

As Wolbachia pipientis is more widely being released into field populations of Aedes aegypti for disease control, the ability to select the appropriate strain for differing environments is increasingly important. A previous study revealed that longer-term quiescence in the egg phase reduced the fertility of mosquitoes, especially those harboring the wAlbB Wolbachia strain. This infertility was also associated with a greater biting rate. Here, we attempt to quantify the effect of this heightened biting behavior on the transmission potential of the dengue virus using a combination of assays for fitness, probing behavior, and vector competence, allowing repeat feeding, and incorporate these effects in a model of R0. We show that Wolbachia-infected infertile mosquitoes are more interested in feeding almost immediately after an initial blood meal relative to wild type and Wolbachia-infected fertile mosquitoes and that these differences continue for up to 8 days over the period we measured. As a result, the infertile Wolbachia mosquitoes have higher virus prevalence and loads than Wolbachia-fertile mosquitoes. We saw limited evidence of Wolbachia-mediated blocking in the disseminated tissue (legs) in terms of prevalence but did see reduced viral loads. Using a previously published estimate of the extrinsic incubation period, we demonstrate that the effect of repeat feeding/infertility is insufficient to overcome the effects of Wolbachia-mediated blocking on R0. These estimates are very conservative, however, and we posit that future studies should empirically measure EIP under a repeat feeding model. Our findings echo previous work where periods of extensive egg quiescence affected the reproductive success of Wolbachia-infected Ae. aegypti. Additionally, we show that increased biting behavior in association with this infertility in Wolbachia-infected mosquitoes may drive greater vector competence. These relationships require further exploration, given their ability to affect the success of field releases of Wolbachia for human disease reduction in drier climates where longer egg quiescence periods are expected.

ASCARIS SUUM EGG RECOVERY FROM SLUDGE SAMPLES AFTER PHASE EXTRACTION.

Some helminth test methods for sanitation samples include a phase extraction step to reduce lipid content and final pellet size before microscopy. Hydrophilic and lipophilic solutions are used to create 2 phases, with a plug of organic material or debris in between, whilst eggs are supposedly compacted at the bottom of the test tube. We tested 10% formalin, acetoacetic buffer, and acid alcohol as the hydrophilic solutions, and ethyl acetate and diethyl ether as the lipophilic solvents for egg recoverability from water, primary sludge, and fatty sludge. Normally, the supernatant and debris plug are discarded and the sedimented pellet of eggs is microscopically examined. We, however, also collected the entire supernatant plus debris plug to determine where eggs were possibly lost. We found that eggs were lost when samples were extracted with 10% formalin + ethyl acetate, 10% formalin + diethyl ether, acetoacetic buffer + ethyl acetate, and acetoacetic buffer + diethyl ether combinations (<50% egg recovery). Acid alcohol + ethyl acetate resulted in 93.2, 89.8, and 57.3% egg recovery in the pellet of water, primary sludge, and fatty sludge, respectively; however, the size of the final pellet was not reduced, defeating the purpose of the extraction step. We thus recommend that this step be excluded.

Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Egg quality in fishes is commonly determined by fertilisation success and cleavage patterns as a phenotypic outcome of underlying regulatory mechanisms. Although these phenotypic estimators of egg quality are useful in farming conditions, these "good quality" egg batches do not always translate to good larval growth and survival. The identification of genes involved in embryonic development may help find links between genetic factors of maternal origin and egg quality. Herein, the relative expression of seven stage-specific developmental genes of Atlantic cod was analysed using quantitative PCR to understand the function during embryogenesis and its relationship with egg quality. Genes ccnb2 and pvalb1 showed significant differential expression between developmental stages and significant upregulation from blastula and somite stages, respectively. The comparison of spawning batches showed that the relative gene expression of genes ccnb2, acta, tnnt3 and pvalb1 was significantly higher from the middle of the spawning season where phenotypic quality estimators establish the best egg quality. Moreover, a positive significant correlation was observed between quality estimators based on egg morphology and the genetic expression of genes acta and acta1 during somitogenesis. This study suggests that the combination of quality estimators, genetics and batch timing could help optimise reproductive protocols for commercial stocks of Atlantic cod.

Beauveria bassiana native strains affect the reproductive index of Rhipicephalus microplus ticks.

Rhipicephalus microplus poses a significant economic threat due to its role in transmitting Babesia bigemina, B. bovis and Anaplasma marginale. Chemical control methods, commonly employed, encounter challenges like resistance, high costs, and environmental concerns. Emerging as an alternative, entomopathogenic fungi, particularly Beauveria bassiana, present a promising avenue for biological control. Molecular identification using the internal transcribed spacer (ITS1-5.8-ITS4) region ensures accurate species identification. This study investigated two B. bassiana strains, assessing their molecular characterization, impact on R. microplus mortality, and reproductive effects on adult females. The Reproductive Aptitude Index (RAI) is employed to evaluate tick egg viability post-treatment, providing insights into the potential of these fungi for tick control. Results indicate the BbLn2021-1 strain causes 96% mortality, and BbSf2021-1 induces 100% mortality. The commercial strain exhibited 28% mortality, while the control treatment showed 12%. Statistical analysis reveals a significant difference between treatments (p < 0.01). The Reproductive Efficiency Index (REI) underscores BbSf2021-1is superiority, yielding lower egg weights than other treatments. Regarding the RAI, BbLn2021-1 and BbSf2021-1 show no significant differences but differ significantly from the commercial and control (p < 0.01). These findings suggest that strains isolated and characterized from the natural environment could have potential applications in field trials, serving as a biocontrol alternative for R. microplus ticks.

Flexible oviposition behavior enabled the evolution of terrestrial reproduction.

Among vertebrates, nearly all oviparous animals are considered to have either obligate aquatic or terrestrial oviposition, with eggs that are specialized for developing in those environments. The terrestrial environment has considerably more oxygen but is dry and thus presents both opportunities and challenges for developing embryos, particularly those adapted for aquatic development. Here, we present evidence from field experiments examining egg-laying behavior, egg size, and egg jelly function of 13 species of Central and South American treefrogs in the genus Dendropsophus, which demonstrates that flexible oviposition (individuals laying eggs both in and out of water) and eggs capable of both aquatic and terrestrial development are the likely factors which enable the transition from aquatic to terrestrial reproduction. Nearly half of the species we studied had previously undescribed degrees of flexible oviposition. Species with obligate terrestrial reproduction have larger eggs than species with aquatic reproduction, and species with flexible reproduction have eggs of intermediate sizes. Obligate terrestrial breeding frogs also have egg masses that absorb water more quickly than those with flexible oviposition. We also examined eight populations of a single species, Dendropsophus ebraccatus, and document substantial intraspecific variation in terrestrial oviposition; populations in rainy, stable climates lay fewer eggs in water than those in drier areas. However, no differences in egg size were found, supporting the idea that the behavioral component of oviposition evolves before other adaptations associated with obligate terrestrial reproduction. Collectively, these data demonstrate the key role that behavior can have in facilitating major evolutionary transitions.

Toxic effects of freshwater grouper (Acrossocheilus fasciatus) eggs on poultry: Morphological and transcriptomic insights into hepatic toxicity.

Fish egg poisoning is a serious and neglected public menace that kills hundreds of people and numerous poultry each year. Freshwater groupers (Acrossocheilus fasciatus) are common food fish in the southeastern regions of China. Their toxic eggs are regarded as a significant public health concern. The molecular mechanisms of egg-toxin toxicity in freshwater grouper to poisoned organisms are elusive. In this study, black-boned chicks were exposed to toxic eggs from freshwater grouper at a lethal dose. The hepatic morphology of the intoxicated chick was assessed. An analysis of the liver gene expression profile was conducted by comparing samples exposed to toxic eggs with control samples using RNA-Seq. The result revealed that an increase in vacuolation and congestion was observed in chicks with toxic eggs exposure. The transcriptome analysis revealed 5421 genes with differential expression, comprising 2810 up-regulated and 2611 down-regulated genes. The genes were primarily linked to energy metabolism, cell apoptosis, cell adhesion, exogenous microbial infection, and cell junction. The most strongly upregulated genes were cholecystokinin (CCK), cholecystokinin A receptor (CCKAR), and unc-80 homolog, NALCN activator (UNC80), and the most downregulated genes were glycine amidinotransferase (GATM), fatty acid desaturase 2 (FADS2), and hexokinase 2 (HKDC1). GO term with the highest enrichment of DEGs is nucleosome assembly. According to KEGG pathways, the three most significant metabolic pathways in the liver are DNA replication, retinol metabolism, and steroid biosynthesis. The results could be crucial for comprehending the negative biological impacts of egg-toxin and its toxic mechanisms. The outcome could provide potential biomarkers of egg-toxin exposure in hepatic, which might be useful for manufacturing an antidote to egg-toxin and providing valuable insights for ecotoxicity studies.

Germline ß-1,3-glucan deposits are required for female gametogenesis in Arabidopsis thaliana.

Correct regulation of intercellular communication is a fundamental requirement for cell differentiation. In Arabidopsis thaliana, the female germline differentiates from a single somatic ovule cell that becomes encased in ß-1,3-glucan, a water insoluble polysaccharide implicated in limiting pathogen invasion, regulating intercellular trafficking in roots, and promoting pollen development. Whether ß-1,3-glucan facilitates germline isolation and development has remained contentious, since limited evidence is available to support a functional role. Here, transcriptional profiling of adjoining germline and somatic cells revealed differences in gene expression related to ß-1,3-glucan metabolism and signalling through intercellular channels (plasmodesmata). Dominant expression of a ß-1,3-glucanase in the female germline transiently perturbed ß-1,3-glucan deposits, allowed intercellular movement of tracer molecules, and led to changes in germline gene expression and histone marks, eventually leading to termination of germline development. Our findings indicate that germline ß-1,3-glucan fulfils a functional role in the ovule by insulating the primary germline cell, and thereby determines the success of downstream female gametogenesis.

Egg multivesicular bodies elicit an LC3-associated phagocytosis-like pathway to degrade paternal mitochondria after fertilization.

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.

Ovicidal activity and cytotoxicity of ethanolic extract of turmeric (Curcuma longa) and green tea (Camellia sinensis) to treat digestive parasite of sheep.

Background: The Trichuris eggs are collected from naturally infected sheep. Natural antihelmintics such as herbal medicines are needed as an alternative, such as natural compounds from endemic plants. Aim: This present study aims to evaluate the ovicidal activity and cytotoxicity effects of ethanolic extract of Curcuma longa (EECL) and Camelia sinensis (EECS) as a biological anthelmintic against the egg of Trichuris sp. Methods: The Trichuris eggs are collected from naturally infected sheep. CMC-Na solution 1% was used as a control. The treatments were 0.12% EECL; 0.24% EECL; 0.15% EECS; 0.30% EECS; a combination of 0.12% EECL and 0.30% EECS; a combination of 0.24% EECL; and 0.15% EECS. Ovicidal activity testing by microscopic examination of eggs treated using different concentrations of EECL extract, EECS, and a combination of them. They were exposed for various times (7, 14, 21, and 28 days) and incubated at room temperature. Results: The study shows that a combination of C. longa extract and tea extract exhibits good ovicidal anthelmintic activity against Trichuris sp. in sheep. Cytotoxicity examination using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) test. Based on MTT data processed using regression analysis, the obtained LC50 from the administration of EECL, EECS, and a combination of both in a ratio of 1:1, 2:2, 1:2, and 2:1. The combination of EECL extract and EECS with the highest concentration produced cell viability of 28.46%, 17.25%, 56.01%, and 46.47%, respectively. Conclusion: It can be concluded that the most cytotoxic ingredient is found in the combination of EECL and EECS (2:2) at 17.25% and the safest is in the ratio (1:2) at 56.01%.

Coevolution between eggs and sperm of insects.

Sexual selection is known to play a major role in the evolution of insect sperm size, whereas natural selection is thought to be a major driver of insect egg size. Despite these differing forms of selection operating, it is possible coevolution between male and female gametes can occur owing to their vital interactions during fertilization. We tested egg-sperm coevolution in insects and found that longer sperm correlated to longer and wider eggs. Moreover, the size of the entry point of sperm into insect eggs (micropyles), was positively related to the diameter of sperm, on average being approximately three times the diameter of the sperm. This suggests a function in reducing and channelling sperm entry, but potentially still leaving space for movement. Our work suggests that greater attention needs to be paid to egg-sperm interactions prior to the point of fertilization as they may influence the evolution of gametes.

Shaping Drosophila eggs: unveiling the roles of Arpc1 and cpb in morphogenesis.

The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this article is to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. We have screened 66 ABPs, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the "capping protein ß" (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.

Egg Mercury Concentration and Egg Size Varies with Position in the Laying Sequence in two Songbird Species.

In birds, mercury embryotoxicity can occur through the transfer of mercury from the female to her eggs. Maternal transfer of mercury can vary by egg position in the laying sequence, with first-laid eggs often exhibiting greater mercury concentrations than subsequently laid eggs. We studied egg mercury concentration, mercury burden (total amount of mercury in the egg), and egg morphometrics by egg position in the laying sequence for two songbirds: tree swallows (Tachycineta bicolor) and house wrens (Troglodytes aedon). Egg mercury concentration in the second egg laid was 14% lower for tree swallows and 6% lower for house wrens in comparison with the first egg laid. These results indicate that in both species, after an initial relatively high transfer of mercury into the first egg laid, a smaller amount of mercury was transferred to the second egg laid. This lower mercury concentration persisted among all subsequently laid eggs (eggs three to eight) in tree swallows (all were 14%-16% lower than egg 1), but mercury concentrations in subsequently laid house wren eggs (eggs three to seven) returned to levels observed in the first egg laid (all were 1% lower to 3% greater than egg 1). Egg size increased with position in the laying sequence in both species; the predicted volume of egg 7 was 5% and 6% greater than that of egg 1 in tree swallows and house wrens, respectively. This change was caused by a significant increase in egg width, but not egg length, with position in the laying sequence. The percentage of decline in mercury concentration with position in the laying sequence was considerably lower in tree swallows and house wrens compared with other bird taxonomic groups, suggesting that there are key differences in the maternal transfer of mercury into songbird eggs compared with other birds. Finally, we performed simulations to evaluate how within-clutch variation in egg mercury concentrations affected estimates of mean mercury concentrations in each clutch and the overall sampled population, which has direct implications for sampling designs. Environ Toxicol Chem 2024;43:1844-1854. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.

Pollination efficiency and the pollen-ovule ratio.

Pollination presents a risky journey for pollen grains. Pollen loss is sometimes thought to favour greater pollen investment to compensate for the inefficiency of transport. Sex allocation theory, to the contrary, has consistently concluded that postdispersal loss should have no selective effect on investment in either sex function. But the intuitively appealing compensation idea continues to be raised despite the lack of theoretical endorsement. We address the theoretical issue with a model that directly represents pollen loss (and ovule loss through floral demise or loss of receptivity) as rate-dependent dynamical processes. These loss rates can be varied to examine the effect of pollination efficiency on optimal sex allocation. Pollen-ovule ratios follow from the sex allocation based on the resource costs of pollen and ovule production. This model confirms conventional findings that pollen loss should have essentially no effect on sexual resource allocation in large, panmictic populations. Pollen limitation of seed set does not alter this conclusion. These results force us to rethink the empirical association of pollination efficiency with low pollen-ovule ratios. This pattern could arise if efficient pollen transport commonly results in stigmatic deposition of cohorts of related pollen. Empirical evidence of correlated paternity supports this explanation.

Evaluation of heating and liming treatments in sand samples artificially contaminated with Ancylostoma spp. eggs.

Ancylostoma spp. are found worldwide. Infected dog and cat feces can contaminate soil in public places. Despite prophylactic measures being available, studies on direct remediation of Ancylostoma-contaminated soils are scarce. This study aimed to determine the impact of heat treatment and liming on the viability of Ancylostoma spp. eggs in artificially contaminated sandy soil. Sterilized sand samples were contaminated with Ancylostoma spp. eggs extracted from infected dogs' feces. Samples were heated (trial I) to 70 °C or 80 °C, then sieved after 24 hours (212, 90, 38, and 25 µm). Larval cultures were assessed for larval development following heat treatment. Five quicklime concentrations (trial II; 50, 30, 20, 10 and 5%) were used to treat sand. The effect of liming on larval cultures was assessed by measuring embryonic development. Filariform larvae were exposed to 20% quicklime (25 °C and 37 °C, 20 min). Heat treatment destroys Ancylostoma spp. eggs and prevents in vitro larval development. Liming at 50, 30, and 20% concentrations made embryonic development impossible. However, filariform larvae treated with 20% lime solution retained their motility. Heating at 70 °C and liming at 20% were sufficient to make Ancylostoma spp. egg embryogenesis impossible in experimentally contaminated sand samples.

An insight into the gut microbiota after Schistosoma japonicum eggs immunization in an experimental ulcerative colitis model.

Schistosome infection and schistosome-derived products have been implicated in the prevention and alleviation of inflammatory bowel disease by manipulating the host immune response, whereas the role of gut microbiota in this protective effect remains poorly understood. In this study, we found that the intraperitoneal immunization with Schistosoma japonicum eggs prior to dextran sulfate sodium (DSS) application significantly ameliorated the symptoms of DSS-induced acute colitis, which was characterized by higher body weight, lower disease activity index score and macroscopic inflammatory scores. We demonstrated that the immunomodulatory effects of S. japonicum eggs were accompanied by an influence on gut microbiota composition, abundance, and diversity, which increased the abundance of genus Turicibacter, family Erysipelotrichaceae, phylum Firmicutes, and decreased the abundance of genus Odoribacter, family Marinifilaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidota. In addition, Lactobacillus was identified as a biomarker that distinguishes healthy control mice from DSS-induced colitis mice. The present study revealed the importance of the gut microbiota in S. japonicum eggs exerting protective effects in an experimental ulcerative colitis (UC) model, providing an alternative strategy for the discovery of UC prevention and treatment drugs.

Assessment of the effects of 2,4-D-dichlorophenoxyacetic acid-based herbicide exposure in eggs and embryos of golden apple snails using OCT.

This study aimed to evaluate the effects of herbicide 2, 4-D-dichlorophenoxy acetic acid on golden apple snail eggs and embryos. Additionally, the study assessed the applicability of optical coherence tomography (OCT), a non-invasive depth cross-sectional microscopic imaging technique, as a novel method, to the best of our knowledge, for studying morphological changes in golden apple snail eggs and embryos, in comparison to the conventional approach of using white light microscopy. The study revealed that the herbicide 2,4-D-dichlorophenoxy acetic acid affected the hatchery rate and morphological changes of the eggs and embryos. The lethal concentration (LC50), representing the concentration of a substance that is expected to cause death in half of the population being studied, of the golden apple eggs and embryos increased with longer exposure time and higher concentrations. The estimated median effective concentration (EC50), which denotes the concentration producing the desired effect in 50% of the exposed golden apple embryos, exhibited a similar trend of change as the LC50. When compared to the microscopic study, it was observed that OCT could be employed to investigate morphological changes of golden apple snail eggs and embryos, enabling evaluation of alterations in both 2D and 3D structures.

Bioactivities of essential oil extracted from Elsholtzia densa Benth. And its main components against Tribolium castaneum eggs and pupae.

This study aimed to develop a relatively natural and safe botanical insecticide for controlling the storage pest Tribolium castaneum in the egg and pupal stages. It examined how Elsholtzia densa Benth. essential oil (EO) and its primary components, ß-caryophyllene and limonene, affected T. castaneum eggs and pupae through contact and fumigation. Among th, the contact activities of ß-caryophyllene against T. castaneum eggs and pupae are LD50 (median lethal dose, 50%) = 0.156 mg/cm2 and ED50 (median effective dose, 50%) = 16.35 mg/pupa respectively. The study also investigated the effect of ß-caryophyllene and limonene on T. castaneum eggs and pupae through synergistic contact and fumigation. When the mixing ratio of ß-caryophyllene and limonene was 7:1, the LD50 value of contact activity against T. castaneum eggs was reduced to 0.100 mg/cm2, displaying an obvious synergistic effect. Experiments were conducted to investigate the antitoxic effect of ß-caryophyllene on T. castaneum eggs and pupae, as well as its effects on the enzymatic activity of acetylcholinesterase, succinate dehydrogenase, glutathione S-transferase and carboxylesterase in T. castaneum pupae. Finally, the molecular docking techniques were employed to confirm the aforementioned effects on enzyme function. The findings of this study might help improve storage pest control with T. castaneum and create eco-friendly insecticides using E. densa EO, ß-caryophyllene, and limonene.

Evaluation of Parasight All-in-One system for the automated enumeration of helminth ova in canine and feline feces.

BACKGROUND:  Digital imaging combined with deep-learning-based computational image analysis is a growing area in medical diagnostics, including parasitology, where a number of automated analytical devices have been developed and are available for use in clinical practice. METHODS: The performance of Parasight All-in-One (AIO), a second-generation device, was evaluated by comparing it to a well-accepted research method (mini-FLOTAC) and to another commercially available test (Imagyst). Fifty-nine canine and feline infected fecal specimens were quantitatively analyzed by all three methods. Since some samples were positive for more than one parasite, the dataset consisted of 48 specimens positive for Ancylostoma spp., 13 for Toxocara spp. and 23 for Trichuris spp. RESULTS: The magnitude of Parasight AIO counts correlated well with those of mini-FLOTAC but not with those of Imagyst. Parasight AIO counted approximately 3.5-fold more ova of Ancylostoma spp. and Trichuris spp. and 4.6-fold more ova of Toxocara spp. than the mini-FLOTAC, and counted 27.9-, 17.1- and 10.2-fold more of these same ova than Imagyst, respectively. These differences translated into differences between the test sensitivities at low egg count levels (< 50 eggs/g), with Parasight AIO > mini-FLOTAC > Imagyst. At higher egg counts Parasight AIO and mini-FLOTAC performed with comparable precision (which was significantly higher that than Imagyst), whereas at lower counts (> 30 eggs/g) Parasight was more precise than both mini-FLOTAC and Imagyst, while the latter two methods did not significantly differ from each other. CONCLUSIONS: In general, Parasight AIO analyses were both more precise and sensitive than mini-FLOTAC and Imagyst and quantitatively correlated well with mini-FLOTAC. While Parasight AIO produced lower raw counts in eggs-per-gram than mini-FLOTAC, these could be corrected using the data generated from these correlations.

Resource allocation strategies and mechanical constraints drive the diversification of stick and leaf insect eggs.

The diversity of insect eggs is astounding but still largely unexplained. Here, we apply phylogenetic analyses to 208 species of stick and leaf insects, coupled with physiological measurements of metabolic rate and water loss on five species, to evaluate classes of factors that may drive egg morphological diversification: life history constraints, material costs, mechanical constraints, and ecological circumstances. We show support for all three classes, but egg size is primarily influenced by female body size and strongly trades off with egg number. Females that lay relatively fewer but larger eggs, which develop more slowly because of disproportionately low metabolic rates, also tend to bury or glue them in specific locations instead of simply dropping them from the foliage (ancestral state). This form of parental care then directly favors relatively elongated eggs, which may facilitate their placement and allow easier passage through the oviducts in slender species. In addition, flightless females display a higher reproductive output and consequently lay relatively more and larger eggs compared with flight-capable females. Surprisingly, local climatic conditions had only weak effects on egg traits. Overall, our results suggest that morphological diversification of stick insect eggs is driven by a complex web of causal relationships among traits, with dominant effects of resource allocation and oviposition strategies, and of mechanical constraints.