An RNA Interference Tool to Silence Genes in Sarcoptes scabiei Eggs

In a quest for new interventions against scabies—a highly significant skin disease of mammals, caused by a parasitic mite Sarcoptes scabiei—we are focusing on finding new intervention targets. RNA interference (RNAi) could be an efficient functional genomics approach to identify such targets. The RNAi pathway is present in S. scabiei and operational in the female adult mite, but other developmental stages have not been assessed. Identifying potential intervention targets in the egg stage is particularly important because current treatments do not kill this latter stage. Here, we established an RNAi tool to silence single-copy genes in S. scabiei eggs. Using sodium hypochlorite pre-treatment, we succeeded in rendering the eggshell permeable to dsRNA without affecting larval hatching. We optimised the treatment of eggs with gene-specific dsRNAs to three single-copy target genes (designated Ss-Cof, Ss-Ddp, and Ss-Nan) which significantly and repeatedly suppressed transcription by ~66.6%, 74.3%, and 84.1%, respectively. Although no phenotypic alterations were detected in dsRNA-treated eggs for Ss-Cof and Ss-Nan, the silencing of Ss-Ddp resulted in a 38% reduction of larval hatching. This RNAi method is expected to provide a useful tool for larger-scale functional genomic investigations for the identification of essential genes as potential drug targets.

Effects of Flow Velocity on the Growth and Survival of Haliotis discus hannai Larvae in the Recirculating Upflow System From the Point of Energy Metabolism

For the abalone Haliotis discus hannai, attachment and metamorphosis are crucial stages in the transition from planktonic to benthic life. Increasing the larval metamorphosis rate by artificially controlling the external environment and simulating natural seawater flow is vital to enhance the hatchery efficiency of H. discus hannai. Thus, in the current study, an upflow recirculating aquaculture unit was designed for the rearing of larval abalone, and the larval hatching rate, survival rate, mode of energy metabolism, and expression levels of metamorphosis-related genes at different flow velocities (0, 5, 10, 20, and 40 L/h) were compared and analyzed. At flow velocities less than 20 L/h, no significant differences occurred in larval hatching, survival, and metamorphosis rates, whereas significant differences were recorded at flow rates of 20 and 40 L/h. Differences were also observed in the activity of enzymes, such as hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH), as well as glycogen levels, at the higher flow rates. These results suggested that velocity in excess of a certain limit leads to a higher glycolysis rate and transition of energy utilization from aerobic to anaerobic metabolism for the abalone larvae. Compared with conventional still-water aquacultural systems, the flow velocity at 5–10 L/h could maintain the water environment stability, and avoid both fertilized eggs from being densely deposited before hatching and the consumption of energy needed to resist high flow velocities. Thus, these results are useful references to enhance the hatchery efficiency, and to conduct large-scale rearing, of abalone larvae.

The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (ΔBbmsn2) and complemented mutant (ΔBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.

Reduced Reproductive Success of Western Baltic Herring (Clupea harengus) as a Response to Warming Winters

Shallow estuaries, bays, and lagoons are generally considered hot spots of ocean productivity that often adjust rapidly to seasonal variations in atmospheric temperatures. During spring when biological reproductive processes begin in the temperate zones, regional climate variability can be immense and uncovering a non-linear biological response, such as fish recruitment to changing temperature regimes might be challenging. Using herring as a paradigm for a response of coastal spring productivity to regional climate drivers, we demonstrated how the annual timing of spawning periods can significantly affect the reproductive success of spring-spawning herring (Clupea harengus) in the western Baltic Sea. An investigation of spawning phenology in consecutive years indicated a temperature threshold range of 3.5–4.5°C triggering initial spawning in the coastal zone. Based on this finding, we analyzed the timing of larval hatching peaks, larval survival and recruitment to the adult population relative to multi-decadal time-series of seasonal sea-surface temperatures. The results revealed that the late seasonal onset of cold periods the corresponding elongation of the period where larvae hatch from the eggs and early larval hatching peaks significantly reduced larval production in a coastal nursery area and finally lead to a reduced abundance of juveniles in the entire distribution area. Using a combination of field research and time series analysis, we presented precedence for shifting regional winter regimes providing a present-day stressor to reproductive capacity of a central component of the coastal food web.

Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel against adult cat flea Ctenocephalides felis and flea egg production in cats

Esafoxolaner, a purified enantiomer of afoxolaner with insecticidal and acaricidal properties, is combined with eprinomectin and praziquantel in NexGard® Combo, a novel topical endectoparasiticide formulation for cats. The efficacy of this novel formulation against adult and immature stages of Ctenocephalides felis fleas was tested in four experimental studies. Two studies were designed to test adulticide efficacy, one to test inhibition of immature stages, and one to test both adulticide efficacy and inhibition of immature stages. In each study, cats were randomly allocated to a placebo control group or to a novel formulation group treated once at the minimum recommended dose. Cats were experimentally infested weekly for one to two months with unfed C. felis originating from North America or Europe. For adulticide efficacy evaluations, live fleas were counted 24 h after treatment and after subsequent weekly infestations. For immature stages, flea eggs were collected and counted weekly for evaluation of egg production inhibition and incubated for larval hatching evaluation. In the three studies testing adult fleas, curative efficacies, 24 h after treatment, were 92.1%, 98.3% and 99.7%; preventive weekly efficacies, 24 h after weekly infestations, remained higher than 95.5% for at least one month. In the two studies testing immature stages, egg production and larval hatching was significantly reduced for at least one month. These studies provide robust evidence of efficacy of the novel formulation against experimental adult flea infestations and for the prevention of environmental contamination by immature flea stages, for at least one month.

CHEMICAL COMPOSITION AND OVICIDA ACTIVITY OF THE ESSENTIAL OIL OF CROTON ARGYROPHYLLUS (EUPHORBIACEAE) LEAVES ABOUT AEDES AEGYPTI (DIPTERA: CULICIDAE

The control of Aedes aegypti, which transmits several arboviruses of importance to public health, has as strategies the use of synthetic insecticides, integrated with environmental control. Its control is mainly directed to the larvae and adult phases. The egg phase is difficult to control, as it has great resistance to environmental conditions. The objective of this work was to evaluate the ovicidal potential of the essential oil obtained from the leaves of Croton argyrophyllus, on Aedes aegypti, as well as to determine the moisture content, the yield, and its chemical composition. The eggs were exposed to the aqueous solution of the essential oil in 10% Tween 80, in the concentration of 12 mg mL-1, for a period of 15, 30, 60, and 120 minutes. Subsequently, the eggs were washed and, after 180 hours of observation, the feed was added. The analysis of the chemical composition of the essential oil was performed by Gas Chromatography coupled to Mass Spectrometry. It was found that up to 180 hours of observation, the highest percentage of larval hatching occurred in eggs exposed to essential oil for 15 min (30%) and the lowest percentage in eggs exposed for 120 min ( less than 10%). After 180 hours, there was an increase in hatching in all treatments. The moisture content was 58.69%, the oil yield was 0.35%, and the main chemical constituents found were bicyclogermacrene and b-cariophylene. It was observed that the essential oil of Croton argyrophyllus, at the concentration evaluated, affects the hatching of Aedes aegypti larvae only in the absence of food.

Priming by Timing: Arabidopsis thaliana Adjusts Its Priming Response to Lepidoptera Eggs to the Time of Larval Hatching

Plants can respond to eggs laid by herbivorous insects on their leaves by preparing (priming) their defense against the hatching larvae. Egg-mediated priming of defense is known for several plant species, including Brassicaceae. However, it is unknown yet for how long the eggs need to remain on a plant until a primed defense state is reached, which is ecologically manifested by reduced performance of the hatching larvae. To address this question, we used Arabidopsis thaliana, which carried eggs of the butterfly Pieris brassicae for 1–6 days prior to exposure to larval feeding. Our results show that larvae gained less biomass the longer the eggs had previously been on the plant. The strongest priming effect was obtained when eggs had been on the plant for 5 or 6 days, i.e., for (almost) the entire development time of the Pieris embryo inside the egg until larval hatching. Transcript levels of priming-responsive genes, levels of jasmonic acid-isoleucine (JA-Ile), and of the egg-inducible phytoalexin camalexin increased with the egg exposure time. Larval performance studies on mutant plants revealed that camalexin is dispensable for anti-herbivore defense against P. brassicae larvae, whereas JA-Ile – in concert with egg-induced salicylic acid (SA) – seems to be important for signaling egg-mediated primed defense. Thus, A. thaliana adjusts the kinetics of its egg-primed response to the time point of larval hatching. Hence, the plant is optimally prepared just in time prior to larval hatching.

Facultative Viviparity in a Flesh Fly (Diptera: Sarcophagidae): Forensic Implications of High Variability in Rates of Oviparity in Blaesoxipha plinthopyga (Diptera: Sarcophagidae)

Flesh flies are major primary consumers of carrion and are commonly found on human remains. Due to this latter feeding habit, their development rates can be used to provide temporal information in forensic investigations. This is usually done by referencing published flesh fly development datasets. Flesh flies are typically assumed to be strictly viviparous and datasets reporting their development rates therefore start at the first larval instar. However, an increasing number of studies has identified oviposition by flesh flies, including the forensically relevant species Blaesoxipha plinthopyga Wiedemann. To assess the impact of egg-laying behavior on casework, oviparity rates and time before larval hatching were assessed under controlled laboratory conditions that reflect common casework conditions in Harris County, Texas. We demonstrated systematic deposition of viable eggs but at a very variable rate between samples. Similarly, the duration between oviposition and larval hatching was highly variable, with some eggs taking more than a day to hatch after deposition. These results highlight the need to account for embryonic development in forensic investigations including B. plinthopyga and advocates for the re-evaluation of the assumed strict viviparity of the Sarcophagidae.

The nonparasitic phase of Dermacentor nitens under field conditions in southeastern Brazil

Dermacentor nitens is a one-host tick that uses domestic equids as main hosts. Herein, the nonparasitic phase of D. nitens was evaluated under field conditions in Pirassununga, São Paulo state, Southeastern Brazil. By exposing engorged females of D. nitens to field conditions (grass plots) for 24 consecutive months, this tick species was able to complete its nonparasitic phase by producing host-seeking larvae in the pasture throughout the year. Preoviposition and egg incubation periods were longer during autumn and winter months than during the other months. The number of larvae generated by engorged females was in most of the times lower during autumn and winter months, as demonstrated by lower egg hatching values. Such conditions could be linked to lower mean temperatures and rainfall. Larvae with the longest longevity hatched from the eggs with the shortest incubation periods. An apparent synchronism of larval hatching during spring was observed from the eggs laid by females during late winter and autumn, which is consistent with the phenomenon of “spring rise”. The results indicate that D. nitens can complete up to five generations per year in southeastern Brazil, providing baseline data to develop future protocols for the appropriate control of D. nitens on horses.

The effect of ivermectin® on fertility, fecundity and mortality of Anopheles arabiensis fed on treated men in Ethiopia

Background Insecticide resistance is a growing threat to malaria vector control. Ivermectin, either administered to humans or animals, may represent an alternate strategy to reduce resistant mosquito populations. The aim of this study was to assess the residual or delayed effect of administering a single oral dose of ivermectin to humans on the survival, fecundity and fertility of Anopheles arabiensis in Ethiopia. Methods Six male volunteers aged 25–40 years (weight range 64–72 kg) were recruited; four of them received a recommended single oral dose of 12 mg ivermectin and the other two individuals were untreated controls. A fully susceptible insectary colony of An. arabiensis was fed on treated and control participants at 1, 4, 7, 10 and 13 days post ivermectin-administration. Daily mosquito mortality was recorded for 5 days. An. arabiensis fecundity and fertility were measured from day 7 post treatment, by dissection to examine the number of eggs per mosquito, and by observing larval hatching rates, respectively. Results Ivermectin treatment induced significantly higher An. arabiensis mortality on days 1 and 4, compared to untreated controls (p = 0.02 and p < 0.001, respectively). However, this effect had declined by day 7, with no significant difference in mortality between treated and control groups (p = 0.06). The mean survival time of mosquitoes fed on day 1 was 2.1 days, while those fed on day 4 survived 4.0 days. Mosquitoes fed on the treatment group at day 7 and 10 produced significantly lower numbers of eggs compared to the untreated controls (p < 0.001 and p = 0.04, respectively). An. arabiensis fed on day 7 on treated men also had lower larval hatching rates than mosquitoes fed on days 10 and 13 (p = 0.003 and p = 0.001, respectively). Conclusion A single oral dose of ivermectin given to humans can induce mortality and reduce survivorship of An. arabiensis for 7 days after treatment. Ivermectin also had a delayed effect on fecundity of An. arabiensis that took bloodmeals from treated individuals on day 7 and 10. Additional studies are warranted using wild, insecticide-resistant mosquito populations, to confirm findings and a phase III evaluation among community members in Ethiopia is needed to determine the impact of ivermectin on malaria transmission.