Control Larvae Research Articles

Histological, histochemical and energy disorders caused by R-limonene on Aedes aegypti L. larvae (Diptera: Culicidae)

The study evaluated the effects of R-limonene, in sublethal concentration, on the histology, histochemistry, biochemistry, and carbohydrates and proteins levels in the third instar Aedes aegypti larvae. The R-limonene (LC50 of 27 ppm) and control groups were analyzed 12 and 24 h after the beginning of treatments. The midgut of the control larvae was composed of cylindrical and elongated cells with a spherical and central nucleus and regenerative cells with a pyramidal shape. After 12 h of treatment, columnar cells, protuberances, and cytoplasmic vacuolization were found. However, 24 h after treatment, complete disorganization of the epithelium was observed. There was a positive reaction in all treatments for the presence of glycogen. However, the midgut of larvae treated with R-limonene showed higher levels. For the total protein, positive marking occurred in all groups evaluated, with higher levels in treatments and the lowest in control 12 h. The levels of total protein and glycogen increased in the treated larvae compared to the 12 h control. Besides, a reduction in total sugar levels was observed in the treated larvae compared to controls 12 and 24 h, being more evident in the last one. Therefore, these results demonstrate that R-Limonene caused pathological changes in the epithelium of the A. aegypti midgut at histophysiological and biochemical levels.

Biodegradation of foam plastics by Zophobas atratus larvae (Coleoptera: Tenebrionidae) associated with changes of gut digestive enzymes activities and microbiome

In order to uncover the plastic types that superworm Zophobas atratus can degrade and the underlying changes associated with plastics consumption, three types of plastics including polystyrene (PS), polyethylene (PE) and polyurethane (PU) foam were used as sole feedstock to feed the superworm larvae for 35 days with bran as control. Compared to the control, PS- or PU-fed larvae showed 100% survival rates, the PE-fed and starvation larvae had decreased survival rates of 81.67% and 65%, respectively. Both plastics-fed and starvation groups showed decreased larvae weight. The consumption rates of PS, PE, and PU were 1.41, 0.30, and 0.74 mg/d/larva, respectively. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and differential scanning calorimeter and thermogravimetric (DSC-TGA) analyses demonstrated the changes of functional groups and thermostability in frass compared to plastic feedstocks, indicating the partial oxidation and degradation of plastics. Among the gut digestive enzymes tested, protease showed increased activities in all plastics-fed groups. Gut microbial communities displayed significant relative abundance changes such as increased abundances of Enterococcus in all plastic-fed groups, Citrobacter in PE-fed group, Dysgonomonas and Sphingobacterium in PS-fed group, and Mangrovibacter in PU-fed group. The latter 3 genera were reported for the first time. In summary, the results demonstrated that Z. atratus could efficiently degrade both PS and PU foam plastics, and the plastic degradation was associated with changes of gut microbial communities and digestive enzyme activities.

Potential use of entomopathogenic nematodes against the soil dwelling stages of onion thrips, Thrips tabaci Lindeman: Laboratory, greenhouse and field trials

Onion thrips Thrips tabaci Lindeman (Thysanoptera: Thripidae) is one of the most damaging insect pests of onion Allium cepa L., which is an economically important agriculture crop cultivated worldwide. This is first comprehensive study in which we have tested a broad range of entomopathogenic nematodes (EPN) species against different developmental stages of T. tabaci in laboratory, greenhouse and field trials. We performed several bioassays to assess the efficacy of different species of EPNs against different soil inhabiting stages of T. tabaci under various conditions. In screening bioassays, at 100 infective juveniles (IJs) cm−2 mortality ranged from 8.5 to 74.2% and 5.0–62.7% among all the tested EPNs (Steinernema carpocapsae (ALL strain), S. glaseri (NC), S. riobrave (355), S. feltiae (SN strain), S. rarum (17 C&E), Heterorhabditis bacteriophora (VS), H. georgiana (Kesha), H. indica (HOM1), H. floridensis (K22) and H. megidis (UK211 strain)) in prepupae and pupae, respectively. Five EPNs species (H. bacteriophora, S. feltiae, S. carpocapsae, S. riobrave and H. indica) selected from the screening bioassay were further evaluated against prepupae, pupae and late second instar larvae at four different concentrations i.e. 50, 100, 150 and 200 IJs cm−2. A clear dose response relationship was observed among all the tested species against all the developmental stages. The prepupal stage was the most susceptible (87.9% mortality) followed by pupae (78.1% mortality) and late second instar larvae (59.4% mortality) when exposed to H. bacteriophora at highest dose tested (200 IJs cm−2). Among the different temperatures tested, maximum efficacy was observed by all the tested EPNs at 25 °C and 30 °C followed by 35 °C and 20 °C with H. bacteriophora and S. feltiae being most virulent among all species at all the tested temperatures. In a potted soil bioassay, the lowest adult emergences were observed in the H. bacteriophora treatment for both prepupae and pupae. In potted plant bioassay under the greenhouse condition, significant lower number of adult emergence was observed among H. bacteriophora as compare to control group. Under field conditions, the lowest number of larvae (4.9 per plant in 2017–18 and 9.6 per plant in 2018–19) and adult (0.1 per plant in 2017–18 and 5.2 per plant in 2018–19) were observed in H. bacteriophora treated plots compared to control larvae (29.4 per plant in 2017–18 and 44.1 per plant in 2018–19) and adults (20.0 per plant in 2017–18 and 28.1 in 2018–19). Our results revealed that EPNs could be included in integrated pest management programs for T. tabaci in onion production systems.

Differential Modulation of the Central and Peripheral Monoaminergic Neurochemicals by Deprenyl in Zebrafish Larvae.

Zebrafish embryos and larvae are vertebrate models increasingly used in translational neuroscience research. Behavioral impairment induced by the exposure to neuroactive or neurotoxic compounds is commonly linked to changes in modulatory neurotransmitters in the brain. Although different analytical methods for determining monoaminergic neurochemicals in zebrafish larvae have been developed, these methods have been used only on whole larvae, as the dissection of the brain of hundreds of larvae is not feasible. This raises a key question: Are the changes in the monoaminergic profile of the whole larvae predictive of the changes in the brain? In this study, the levels of ten monoaminergic neurotransmitters were determined in the head, trunk, and the whole body of zebrafish larvae in a control group and in those treated for 24 h with 5 M deprenyl, a prototypic monoamine-oxidase B inhibitor, eight days post-fertilization. In control larvae, most of the monoaminergic neurochemicals were found at higher levels in the head than in the trunk. Significant changes were found in the distribution of some neurochemicals after deprenyl-treatment, with serotonin and norepinephrine increasing in both the head and the trunk, whereas dopamine, L-DOPA, and homovanillic acid levels were only modulated in the head. In fact, the highly significant increase in dopamine levels observed in the head after deprenyl-treatment was not detected in the whole-body analysis. These results indicate that the analysis of neurotransmitters in the zebrafish larvae whole-body should not be used as a general surrogate of the brain.

Infection with Borrelia afzelii and manipulation of the egg surface microbiota have no effect on the fitness of immature Ixodes ricinus ticks

Arthropod vectors carry vector-borne pathogens that cause infectious disease in vertebrate hosts, and arthropod-associated microbiota, which consists of non-pathogenic microorganisms. Vector-borne pathogens and the microbiota can both influence the fitness of their arthropod vectors, and hence the epidemiology of vector-borne diseases. The bacterium Borrelia afzelii, which causes Lyme borreliosis in Europe, is transmitted among vertebrate reservoir hosts by Ixodes ricinus ticks, which also harbour a diverse microbiota of non-pathogenic bacteria. The purpose of this controlled study was to test whether B. afzelii and the tick-associated microbiota influence the fitness of I. ricinus. Eggs obtained from field-collected adult female ticks were surface sterilized (with bleach and ethanol), which reduced the abundance of the bacterial microbiota in the hatched I. ricinus larvae by 28-fold compared to larvae that hatched from control eggs washed with water. The dysbiosed and control larvae were subsequently fed on B. afzelii-infected or uninfected control mice, and the engorged larvae were left to moult into nymphs under laboratory conditions. I. ricinus larvae that fed on B. afzelii-infected mice had a significantly faster larva-to-nymph moulting time compared to larvae that fed on uninfected control mice, but the effect was small (2.4% reduction) and unlikely to be biologically significant. We found no evidence that B. afzelii infection or reduction of the larval microbiota influenced the four other life history traits of the immature I. ricinus ticks, which included engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. A retrospective power analysis found that our sampling effort had sufficient power (> 80%) to detect small effects (differences of 5% to 10%) of our treatments. Under the environmental conditions of this study, we conclude that B. afzelii and the egg surface microbiota had no meaningful effects on tick fitness and hence on the R0 of Lyme borreliosis.

Study on the implementation of different biofloc sedimentable solids in improving the water quality and survival rate of mud crab, Scylla paramamosain larvae culture

Microbial communities in biofloc technology (BFT) are responsible in minimalize water exchange and producing microbial proteins that are beneficial as a supplementals diet for mud crab, Scylla paramamosain larvae culture. Therefore, a study was conducted to determine the effect of bacterial communities in different sedimentable solids in order to improve the water quality and survival rate of S. paramamosain larvae culture. The results successfully identified that the number of pathogenic bacteria, Vibrio spp. decreased when the heterotrophic bacteria thrived in treatment tank with 4 ml/L sedimentable solids. The group of heterotrophic bacteria were able to adapt in developing their population while effectively uptake the ammonium and nitrite components. The ammonium and nitrite level in treatment tank depleting at an early stage of the experiment, and constantly low until the end of experiments (ammonium <0.20 mg/L, nitrite <0.10 mg/L). The survival rates of S. paramamosain larvae in both control and treatment tanks were identified <2.0%, however, sedimentable solids of 2 ml/L was identified suitable to be applied in crab larvae culture as it can help to reach megalopa stages within 17 days of culture period. Overall, it can be concluded that bacteria communities in biofloc are able to regulate nutrients load and maintaining water quality and also help in increased survival rate and development performance in the low sedimentable solids of biofloc culture treatment. Therefore, sedimentable solids of 2 ml/L is suggested to be potentially applied in S. paramamosain larvae cultures from this research finding.

Precocious Metamorphosis of Silkworm Larvae Infected by BmNPV in the Latter Half of the Fifth Instar.

The mulberry silkworm (Bombyx mori) is a model organism, and BmNPV is a typical baculovirus. Together, these organisms form a useful model to investigate host–baculovirus interactions. Prothoracic glands (PGs) are also model organs, used to investigate the regulatory effect of synthetic ecdysone on insect growth and development. In this study, day-4 fifth instar silkworm larvae were infected with BmNPV. Wandering silkworms appeared in the infected groups 12 h earlier than in the control groups, and the ecdysone titer in infected larvae was significantly higher than that of the control larvae. We then used RNA sequencing (RNA-seq) to analyze silkworm PGs 48 h after BmNPV infection. We identified 15 differentially expressed genes (DEGs) that were classified as mainly being involved in metabolic processes and pathways. All 15 DEGs were expressed in the PGs, of which Novel01674, BmJing, and BmAryl were specifically expressed in the PGs. The transcripts of BmNGDN, BmTrypsin-1, BmACSS3, and BmJing were significantly increased, and BmPyd3, BmTitin, BmIGc2, Novel01674, and BmAryl were significantly decreased from 24 to 72 h in the PGs after BmNPV infection. The changes in the transcription of these nine genes were generally consistent with the transcriptome data. The upregulation of BmTrypsin-1 and BmACSS3 indicate that these DEGs may be involved in the maturation process in the latter half of the fifth instar of silkworm larvae. These findings further our understanding of silkworm larval development, the interaction between BmNPV infection and the host developmental response, and host–baculovirus interactions in general.

Developing an ACURE to Investigate the Effects of Manganese on Physiological Processes

To engage freshman college students in research and STEM, we will utilize an authentic course-based undergraduate research experience (ACURE) approach through a focused research project. Introducing students to novel research projects within a course setting provides a structured way to expose participants to the process of scientific research. Involvement in the design and implementation of an experiential paradigm promotes creativity among students and encourages them to become more engaged. Through the process of gathering and analyzing data, students will gain a realistic perspective of scientific research. For this class project, we chose to focus on studying the effects of manganese on behavior, neural function and cardiovascular physiology of Drosophila melanogaster larvae and crayfish models. Emerging research has repeatedly demonstrated that overexposure to manganese in humans can lead to acute cardiovascular disorders and a series of symptoms manifesting in a manner similar to that of Parkinson's disease (PD). We have designed the class in order to gain a multifaceted understanding of these effects. To do so, the students will first examine larval crawling rate, mouth hook movement rate, and touch sensitivity of control larvae and larvae exposed to a maximum non-lethal dose of manganese in order to analyze the effect that manganese has on the behavior of D. melanogaster. Then, students will use optogenetics to activate and inactivate dopaminergic neurons in order to compare behavioral responses of the control group to the group exposed to manganese. In doing so, they will be able to gain understanding of another method of scientific investigation and further build upon the variety of data found throughout the study. Manganese also has the potential to block voltage-gated calcium channels of the heart, so the students will additionally study the effect of manganese exposure on D. melanogaster heart rate and rhythmicity in order to demonstrate the effect that manganese can have on other areas of the body. Finally, students will study the synaptic transmission at glutamatergic neuromuscular junctions by observing and recording data from the muscle fibers of both larval D. melanogaster and crayfish. Like the heart, these neuromuscular junctions contain voltage-gated calcium channels, so the students will be able to further explore the various effects of manganese exposure. The utilization of crayfish in this study will allow the students to gain a new perspective of the effect of manganese because of the ability to record crayfish action potentials intracellularly, something that is not possible to study in D. melanogaster. Overall, we believe that this student-driven research approach will promote student engagement and foster a deeper understanding of the scientific process while also widening the breadth of knowledge in the research field.

Investigating Acute and Subchronic Effects of Neonicotinoids on Northwestern Salamander Larvae

This research investigated the adverse effects of neonicotinoids on the Northwestern salamander (Ambystoma gracile; NWS) after acute and subchronic exposures during early aquatic life stages via whole organism (i.e., growth, development) and molecular (i.e., gene expression) level endpoints. In a 96-h exposure, NWS larvae were exposed to four imidacloprid concentrations (250, 750, 2250, 6750µg/L) and a water control treatment, and no effects on survival, body weight, snout-vent length (SVL), and total body length were observed. However, a significant 1.70- and 2.33-fold decrease in thyroid receptor β (TRβ) mRNA expression levels were detected in the larvae exposed to 750 and 2250µg/L imidacloprid, respectively, compared with the larvae in the water control. In subsequent subchronic experiments, NWS larvae were exposed for 35days to imidacloprid alone and an equal part mixture of neonicotinoids (imidacloprid, clothianidin, and thiamethoxam (ICT)) at three concentrations (10, 100 and 1000µg total neonicotinoids/L) and a water control. In these experiments, there were no effects on larval survival, body weight, SVL, and total body length. However, advanced development of larvae in the 100µg/L imidacloprid treatment was observed compared with the control after 35-day imidacloprid exposure, providing some evidence of disruption of the thyroid endocrine axis at an environmentally relevant concentration. Ultimately, there is a paucity of studies conducted examining the sensitivity of salamanders to pollutants; thus, this study reports novel findings that will contribute to understanding the sensitivity of a Caudate amphibian model to a common environmental pollutant.

Activity of Some Plant and Fungal Metabolites towards Aedes albopictus (Diptera, Culicidae).

Aedes albopictus (Skuse) is a widespread mosquito, a vector of important human arboviruses, including Chikungunya, Dengue and Zika. It is an extremely difficult species to control even for the onset of resistances to chemicals insecticides, therefore ecofriendly products are urgently needed. In this study, the activity of Amaryllidaceae alkaloids and some of their semisynthetic derivatives, of 2-methoxy-1,4-naphthoquinone and two analogues, of cyclopaldic acid and epi-epoformin on the survival and development of Ae. albopictus larvae was evaluated. First-instar larval exposure for 24 and 48 h to cyclopaldic acid, resulted in mortality mean per-centage of 82.4 and 96.9 respectively; 1,2-O,O-diacetyllycorine 48h post-treatment caused 84.7% mortality. Larval and pupal duration were proved to decrease significantly when larvae were exposed to cyclopaldic acid, 1,2-O,O-diacetyllycorine and N-methyllycorine iodide. The mean number of third-instar larvae surviving to 2-methyl-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone and 2-methoxy-1,4-naphthoquinone was significantly lower than the number of correspondent control larvae over the time. This study indicated that 1,2-O,O’-diacetyllycorine, N-methyllycorine iodide, cyclopaldic acid and 1,4-naphthoquinone structural derivatives have good potential for developing bioinsecticides for mosquito control programs. The obtained results are of general interest due to the global importance of the seri-ous human diseases such a vector is able to spread.

Experimental simulations of climate change induced mismatch in oak and larval development rates impact indicators of fitness in a declining woodland moth

Consequences of climate change‐driven shifts in the relative timing of spring activities of interacting species are insufficiently understood, especially for insects. We use a controlled experiment which simulates a trophic mismatch scenario in which lepidopteran larvae predominately feed on older leaves due to foliage developing faster than larvae growth rates. As a case study our experiment uses Orthosia cerasi, which is a widespread but declining woodland moth whose UK declines appear to be driven by warming temperatures. In the control experiment larvae are fed young oak Quercus robur leaves (bud burst stages six and seven), whilst in the treatment newly emerged larvae are fed young leaves but then gradually transition to feed on older leaves (post bud burst stage seven). We assess impacts on duration of the larval stage, pupal size and overwintering duration and survival. Larvae in the phenological mismatch treatment had a longer larval period, and smaller and lighter pupae. Larval diet did not carry over to influence emergence dates as earlier pupation of control larvae was balanced by an equivalent increase in the duration of the pupal stage. Increased time spent as larvae could increase predation rates from avian predators, whilst slowing the seasonal decline in food availability for those bird species. Reduced pupal size and weight are indicators of lower fecundity in emerging adults. Notably, we find that adults emerging from the mismatch treatment exhibited greater rates of abnormal vestigial wing development, which is likely to further reduce fitness. Trophic mismatches in which caterpillars have reduced availability of young leaves may thus contribute to the population declines observed in many woodland moth species due to increased mortality at larval stages, and adverse effects of early life conditions that reduce the reproductive success of emerging adults.

CRISPR/Cas9 mutagenesis reveals a role for ABCB1 in gut immune responses to Vibrio diazotrophicus in sea urchin larvae.

The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host-microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment.

Uhrf1 and dnmt1 Loss Induces an Immune Response in Zebrafish Livers Due to Viral Mimicry by Transposable Elements.

Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, uhrf1 and dnmt1. We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify tnfa as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of tnfa activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors sting and mavs reduced expression of interferon response genes and tnfa depletion dramatically reduced cell death in uhrf1 mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that tnfa mediates cell death as a potential mechanism to eliminate these damaged cells.

Cottonseed Press Cake as a Potential Diet for Industrially Farmed Black Soldier Fly Larvae Triggers Adaptations of Their Bacterial and Fungal Gut Microbiota.

Black soldier fly larvae (Hermetia illucens, Diptera: Stratiomyidae) are used for the bioconversion of organic side products into valuable compounds such as proteins, lipids and chitin. However, the economic competitiveness of farmed insects compared to conventional protein production systems in agriculture and aquaculture depends on the availability of large quantities of inexpensive insect feed. Cottonseed press cake (CPC) is a side-stream of cotton production that is rich in proteins and lipids but unsuitable as feed for several farmed animals, except ruminants, due to the presence of the anti-nutritional sesquiterpenoid gossypol. Here, we tested CPC as a feed for black soldier fly larvae and studied the impact of this diet on the gut microbiome. Larvae reared on CPC developed normally and even showed a shorter life-cycle, but were smaller at the end of larval development than control larvae reared on chicken feed. The adaptability of the larvae to different diets is mediated by their versatile gut microbiome, which facilitates digestion and detoxification. We therefore used amplicon sequencing to analyze the bacterial and fungal communities associated with larvae reared on each diet, revealing differences between the larval guts and frass (residual feed substrate) as well as differences between the two diet groups. For example, Actinomycetaceae and Aspergillaceae were significantly enriched in guts of the CPC diet group and may help to metabolize compounds such as gossypol. Potentially probiotic yeasts and beneficial Enterobacteriaceae, which presumably belong to the core microbiota, were detected in high relative abundance in the gut and frass, indicating a functional role of these microbes, especially the protection against pathogens. We conclude that CPC may be suitable as an inexpensive and environmentally sustainable feed for the industrial rearing of black soldier flies.

Immunological interactions of Chilo suppressalis Walker (Lepidoptera: Crambidae) with the native entomopathogenic fungi

Entomopathogenic fungi can attack many insect hosts and have been applied as the eco-friendly alternatives to synthetic chemicals for the control of pests. Insects have developed different defense systems encountering entomopathogens including humoral and cellular immune responses. In the present study, injection of some native entomopathogenic fungi to the Chilo suppressalis Walker larvae resulted in an enhancement of the cellular and antimicrobial defenses. The numbers of total and differential hemocytes increased rapidly in the first 3 and 6 h but those gradually reduced 12 and 24 h post-injections. The nodule formation and phenoloxidase activity increased at the time intervals after fungal infection. A similar trend was found in the transcription of antimicrobial peptides including attacin1 and 2, cecropin1 and 2, gallerimycin, defensin, lysozyme, and prophenoloxidase-activating proteinase-3 during infection fungi. In all cases, the target gene transcription was upper in the larvae injected by the fungi than that of control larvae. These results may elucidate better knowledge on the interaction of the fungi present in agroecosystems with the target insect pest.

Zebrafish (Danio rerio) larvae show behavioral and embryonic development defects when exposed to opioids at embryo stage

Opioid abuse continues to plague society, and in recent years, there has been an epidemic, leading to increased addiction and death. It is poorly understood how prenatal opioid use affects the lives of children. The aim of this work was to evaluate the effect of early embryonic codeine or morphine exposure in zebrafish (Danio rerio), examining gastrulation progression (epiboly), teratogenic effects, mortality and locomotor behavior response to light/dark cycles. Zebrafish embryos were exposed to codeine or morphine (designated C or M) at 1, 5 or 10 mg/L (designated 01, 05 or 10, respectively) from 3 to 24 h postfertilization (hpf) or from 3 to 48 hpf (designated −24 or − 48 for 1 or 2 days of exposure, respectively). The C10–24, C01–48, C05–48 and C10–48 groups showed significantly smaller eyes than control larvae at 7 days postfertilization (dpf). Locomotor behavior of control larvae in light/dark cycles showed greater swimming time and distance in dark cycles. Two-day codeine exposure produced strong effects, showing no significant response due to light/dark cycles in distance moved. Morphine exposed groups showed similar effects as observed in 2-day codeine exposed groups, showing less large movement activity and also no significant difference between inactive duration in response to light/dark cycles. In conclusion, we observed low teratogenic effects and mortality effects. Animals exposed to high levels and higher exposure times of opioids were hypoactive, relative to controls, in the dark period. Future studies will be needed to understand the neural defects producing behavior changes.

Metabolomic Profiling Unveils the Impact of Non-Doped and Heteroatom-Doped Carbon Nanodots on Zebrafish (Danio rerio) Embryos.

Recently, concern has been raised over the transport, transformation, and fate of carbon nanodots (CNDs) after their release into the environment. Their toxicity towards organisms and humans has recently been addressed as an important issue. In this study, a metabolomic approach was employed to obtain an insight into the effect of CNDs (either pristine or doped with nitrogen and nitrogen/sulfur) on zebrafish. Embryos were exposed to concentrations corresponding to lethal concentration (LC) LC50 (550, 400, and 150 μg mL−1), LC50/2 (275, 200, and 75 μg mL−1), and LC50/4 (138, 100, and 38 μg mL−1) of the three CNDs (non-doped, N-doped, and N,S-codoped, respectively) to scrutinize the interactions of the CNDs with the larvae. Numerous differences in the metabolic pathways were recorded in all cases. Seven metabolic pathways were detected in the control larvae. When the larvae were exposed to concentrations equal to LC50, LC50/2, and LC50/4 of non-doped CNDs, 12, 12, and 3 metabolic pathways were detected, respectively. In the case of N-doped CNDs, 4, 7, and 4 pathways were detected, while in the case of N,S-codoped CNDs, 8, 5, and 5 pathways were detected when exposed to concentrations of LC50, LC50/2, and LC50/4, respectively. In all cases, certain metabolic pathways were altered while others were either down-regulated or up-regulated. Some of these changes include the activation of alanine, aspartate, and glutamate metabolism, aminoacyl-tRNA biosynthesis, butanoate metabolism, D-glutamine, and D-glutamate metabolism, glutathione metabolism, selenoamino acid metabolism, valine, leucine, and isoleucine degradation pathways. Moreover, the deactivation of starch and sucrose metabolism, the glycine, serine, and threonine metabolism, among others, were recorded. Our findings underline the importance to further study the impact of CNDs on marine organisms. As zebrafish has been shown to share many similarities with humans in bioprocesses and genome, it can be assumed that CNDs may also pose a threat to human health.

Functional identification of an opsin kinase underlying inactivation of the pineal bistable opsin parapinopsin in zebrafish

In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency.

The development of the solitary bee Osmia bicornis is affected by some insecticide agrochemicals at environmentally relevant concentrations

Solitary bees provide essential pollination services for many arable crops, but are prone to global decline. Agricultural intensification, which is connected with pesticide usage, is among major threats to bees and, thus, to the food security and ecosystem stability. As it may not be possible to cease pesticide usage currently because of the growing demand for food, it is crucial to understand the pesticide toxicities to bees for better protection of pollinator populations. The majority of studies have focused on social bees, and those on solitary bees studied effects of adult exposure, whereas these bees are also likely to be exposed as larvae via the consumption of contaminated pollen. Here, the effects of three commonly used insecticide-based plant protection products on the development of the solitary bee, Osmia bicornis (red mason bee), were studied by exposing larvae to insecticide-contaminated multifloral pollen. The tested insecticides were: Dursban480EC, containing the organophosphate chlorpyrifos (CHP), Sherpa100EC, containing the pyrethroid cypermethrin (CYP), and Mospilan20SP with the neonicotinoid acetamiprid (ACT). When compared to the control larvae fed with uncontaminated-pollen, both CHP and CYP significantly reduced the O. bicornis larval survival and their body mass at all tested concentrations. In contrast, ACT did not affect either larval survival or body mass, but the length of larval stage to cocoon formation was significantly shortened compared to controls. None of studied insecticides affected the mass of cocooned individuals. However, at least 80% of individuals exposed to any of the tested insecticides died before reaching the adult stage, whereas 43% of the controls emerged successfully after overwintering. Although no clear monotonic dose-response relationships were found, our study showed that at least some insecticide formulations affect the development of O. bicornis even at concentrations actually found in pollen in the field, indicating an urgent need for revising current pesticide usage recommendations.

Chemical characterization of billy goat weed extracts Ageratum conyzoides (Asteraceae) and their mosquitocidal activity against three blood-sucking pests and their non-toxicity against aquatic predators.

The petroleum ether crude extracts of A. conyzoides (Pe-Ac) were used to treat three medically intimidating pests of Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, to evaluate their non-target screening against the mosquito predator. The chemical scanning of Pe-Ac through GC-MS analysis revealed a total of nine compounds and the maximum peak area was observed in 1,5-Heptadien-3-yne (22.14%). At the maximum dosage of Pe-Ac (200ppm), significant larvicidal activity was shown against the fourth instars of Ae. aegypti (96%), An. stephensi (93%), and Cx. quinquefasciatus (92%) respectively. The percentages of oviposition deterrence index (ODI) of all three mosquito vectors are maximum at the highest sub-lethal dosage of Pe-Ac (75ppm) and minimum at the control dosage. The sub-lethal dosage blocked the activity of carboxylesterase activity and upregulated the detoxifying enzyme activity in a dose-dependent way. The adulticidal activity of Pe-Ac showed that the maximum adult mortality rate (100%) was recorded at the prominent dosage of Pe-Ac 600ppm against the vectors of all three mosquitos at the maximum adulticidal time of 30min. Histopathological investigation of fourth instar larvae of all three mosquitos treated with a sub-lethal dosage of Pe-Ac showed that the midgut cells (epithelium, lumen, and peritrophic matrix) are ruptured completely whereas they appear to be normal in control larvae. The non-toxicity evaluation of Pe-Ac compared with the chemical toxin Temephos in aquatic predator Toxorhynchites splendens revealed that the plant extracts are harmless even at the prominent dosage (1000ppm) as compared to Temephos (1 and 2ppm) and displayed a higher mortality rate against the mosquito predators. Thus the safety index recommends that the Pe-Ac is more explicit to targets and a suitable auxiliary to chemical pesticides.