Third-instar Larvae Research Articles

Dual actions of certain essential oils in combating mosquito Culex pipiens (Diptera: Culicidae)

Author : Ahmed S. Hashem, Ahmed H. Abdelwahab, Mohamed A.M. Shahat, Marwa M. Ramadan Mosquitoes are one of the most annoying and potentially dangerous insects on the planet as they are infamous for transmitting diseases such as malaria, dengue fever, Zika virus, West Nile virus, and many others. In order to protect public health and stop the spread of diseases like dengue, zika, and malaria, mosquito control is essential. The current study attempts to evaluate the toxicity and repellant qualities of lavender camphor, and garlic, essential oils against Culex pipiens. After 72 hours, the essential oil demonstrated good larvicidal action in a lab setting against third-instar larvae (LC50 = 341.12, 430.79, and 624.15 ppm, respectively). At all dosages, lavender oil offers a comprehensive repellant that endures longer. In contrast, there were no appreciable variations in the repellents' duration of protection against C. pipiens. These findings provide insight into potential use of essential oils of garlic, camphor, and lavender for the management of mosquitoes.

Nematodes and acanthocephalans of hygienic-sanitary importance parasitizing Hyporthodus niveatus (Valenciennes, 1828) (Actinopterygii) collected from fish markets of the municipality of Niterói, RJ, Brazil

Hyporthodus niveatus is an appreciated and valuable commercial fish species in the municipality of Niterói, state of Rio de Janeiro, Brazil, due its excellent quality flesh. The constant presence of helminths in the abdominal musculature, viscera and serosa of individuals of the species has been the subject of complaints among local fish traders because of economic losses due to their repugnant aspect. Considering their hygienic-sanitary importance and significance for collective health, the presence of helminths was investigated in 20 individual fish of H. niveatus purchased from fish markets in the municipality of Niterói in 2021-2022. Nematodes, identified as third-instar larvae of Contracaecum sp., Terranova sp., Hysterothylacium deardorffoverstreetorum and Raphidascaris sp., were found parasitizing the intestine and abdominal cavity. Acanthocephalans, identified as juveniles of Corynosoma australe, were found parasitizing the intestine. The highest parasitic indices were for H. deardorffoverstreetorum, with prevalence of 30%, mean intensity of 1.5, mean abundance of 0.45 and infection ranges of 1-2. Considerations about the zoonotic potential and hygienic-sanitary significance of these parasites are presented in order to increase food safety for consumers.

Resistance of Third and Fifth Instars of Codling Moth, Cydia pomonella (L.) to the Insecticide Deltamethrin in As-Suwayda Governorate, Syria

Bou Hamdan, R., W. Kassis and M. Bufaur. 2023. Resistance of Third and Fifth Instars of Codling Moth, Cydia pomonella (L.) to the Insecticide Deltamethrin in As-Suwayda Governorate, Syria. Arab Journal of Plant Protection, 41(2): 183-189. https://doi.org/10.22268/AJPP-41.2.183189 Codling moth, Cydia pomonella (L.) is a key pest of global importance that affects apple fruit production and its populations have developed resistance to insecticides in many apple production areas. Third-instar larvae of Cydia pomonella has developed a vigor tolerance to deltamethrin in the fields of As-Suwayda Governorate, Syria. The resistance rate (RR) was (5, 5.6, 6.79) after (24, 48, 72) hours after treatment with deltamethrin, respectively, while ultra-vigor tolerance was observed in the non-diapausing fifth-instar larvae of codling moth (males and females), the resistance rate (RR) was (6.42, 7.75, 9.67) for males and (8.49, 9.84, 10.26) for females after (24, 48, 72) hours, 188 مجلة وقاية النبات العربية، مجلد ،41 عدد 2 ) 2023( respectively, It was noted that females of the non-diapausing fifth-instar larvae was more resistant than males. Based on the results, it is recommended to exclude deltamethrin for a while, using it with other insecticides, not re-using it many times or mixing it with one of the synergists. Keywords: Cydia pomonella (L.), non-diapausing larvae, insecticide resistance, resistance rate, deltamethrin

Protective effect of the newly synthesized and characterized charge transfer (CT) complex against arecoline induced toxicity in third-instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9: experimental and theoretical mechanistic insights.

Agents that suppress the toxic effect of arecoline (a chemical present in the Areca nut fruit) have become a need of the hour owing to its several harmful effects on human beings. Although some drug molecules have been developed for this purpose, yet, simple, easy to prepare, and economical molecules with remarkable potency are still a challenge to design. The present work thus becomes important as it involves the synthesis of a new charge transfer complex (CTC) material, which has, for the first time, been screened to investigate its effect on the toxic effects of arecoline. The newly designed material (CL), which is generated from the reaction between 2,4,6-trinitrophenol (TNP) and pyrazole (PYZ), has been crystallized by a slow evaporation method and characterized by employing spectral studies including single crystal X-ray crystallography. Spectrophotometry studies with the inclusion of the Benesi-Hildebrand equation reveal 1 : 1 stoichiometry and physical parameters of CL. Assays were used for determining the protective effect of CL against arecoline. CL was found to (dose-dependently) decrease β-galactosidase activity, damage in tissue and DNA damage caused by arecoline (80 μM) in the third-instar larvae of the transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. The possible mechanism of this effect was explored through fluorescence and UV-vis spectroscopy. The possibility of suppression of arecoline action on the muscarinic acetylcholine receptor 1-G11 protein complex (found in the cell membrane) in the presence of CL was studied theoretically by molecular docking. Density functional theory (DFT) also theoretically supported various aspects of the designed material concerning the energy profile of the orbitals (HOMO-LUMO) as well as the energy minimized structure. Furthermore, time dependent (TD) DFT corroborated the electronic properties of the designed material.

Ionizing radiation alters functional neurotransmission in Drosophila larvae.

Patients undergoing cranial ionizing radiation therapy for brain malignancies are at increased risk of long-term neurocognitive decline, which is poorly understood and currently untreatable. Although the molecular pathogenesis has been intensively researched in many organisms, whether and how ionizing radiation alters functional neurotransmission remains unknown. This is the first study addressing physiological changes in neurotransmission after ionizing radiation exposure. To elucidate the cellular mechanisms of radiation damage, using calcium imaging, we analyzed the effects of ionizing radiation on the neurotransmitter-evoked responses of prothoracicotropic hormone (PTTH)-releasing neurons in Drosophila larvae, which play essential roles in normal larval development. The neurotransmitters dopamine and tyramine decreased intracellular calcium levels of PTTH neurons in a dose-dependent manner. In gamma irradiated third-instar larvae, a dose of 25 Gy increased the sensitivity of PTTH neurons to dopamine and tyramine, and delayed development, possibly in response to abnormal functional neurotransmission. This irradiation level did not affect the viability and arborization of PTTH neurons and successful survival to adulthood. Exposure to a 40-Gy dose of gamma irradiation decreased the neurotransmitter sensitivity, physiological viability and axo-dendritic length of PTTH neurons. These serious damages led to substantial developmental delays and a precipitous reduction in the percentage of larvae that survived to adulthood. Our results demonstrate that gamma irradiation alters neurotransmitter-evoked responses, indicating synapses are vulnerable targets of ionizing radiation. The current study provides new insights into ionizing radiation-induced disruption of physiological neurotransmitter signaling, which should be considered in preventive therapeutic interventions to reduce risks of neurological deficits after photon therapy.

Description of the mature larva of Suphisellus curtus (Sharp, 1882) (Coleoptera: Noteridae) with chaetotaxy analysis

The third-instar larva of Suphisellus curtus (Sharp, 1882) is described and illustrated for the first time, including morphometric and chaetotaxic analyses of the cephalic capsule, head appendages, legs, last abdominal segment, and urogomphus. This is the second detailed larval description for the genus Suphisellus Crotch, 1873. Larvae of this genus can be readily separated from other known noterid larvae by the strongly developed galea, subequal in length to maxillary palpus. Suphisellus curtus differs from S. rufipes (Sharp, 1882) in the dorsal colour pattern, S. rufipes being entirely testaceous and S. curtus testaceous with transverse brown bands between body segments.

Potential of neem oil extract® against Palmetto weevil larvae, <em>Rhynchophorus cruentatus </em>Fabricius (Coleoptera: Curculionidae) and its impact on some detoxification enzymes

Palmetto weevil, Rhynchophorus cruentatus (Fabricius) (Coleoptera: Curculionidae), is considered the giant weevil in North America (Weissling & Giblin-Davis, 1997). It is a severe pest of palm trees, especially cabbage palms (Sabal palmetto). Larval stages feed heavily on the internal palm soft tissues causing total palm loss (Hunsberger et al., 2000). We know that reports about controlling this pest are very scarce. This research project focuses, for the first time, on controlling Rhynchophorus cruentatus via plant-based insecticides. This study examined the potential of using neem oil extract®, a commercial product, against the third-instar larvae of palmetto weevil (R. cruentatus). Impacts of neem oil extract® on mortality, larval weight, the activity of detoxification enzymes, and thei gene expression levels were examined. Neem oil extract® manifested dose-dependent larvicidal activity against the third-instar larvae of R. cruentatus. Investigations revealed higher mortality and reduction in weight 24 hours post-treatment. LC50 and LC90 values were estimated 24 h post-treatment to be 12.04% and 26.48%, respectively. Biochemical analysis revealed increasing activities of three detoxification enzymes (Acetylcholinesterase, Glutathione S-transferase, and Superoxide dismutase) in the third-instar larvae after 8 h of treatment with LC50. A significant elevation in the expression levels of detoxification genes (Acetylcholinesterase, Glutathione S-transferase, Cytochrome P450, and Superoxide dismutase) was recorded in the treated larva. Our findings help to underline the detoxification mechanisms of R. cruentatus larva against neem oil extract® at both biochemical and molecular levels. Thus, neem oil extract® had a lethal potential against third-instar larvae of R. cruentatus and is suggested as a safe bioinsecticide that may be used in IPM of palm trees as an alternative to chemical insecticides.

Identification, Pathogenicity, and Culture Conditions of a New Isolate of Cordyceps javanica (Hypocreales: Cordycipitaceae) From Soil.

This study decribes a highly effective insecticidal isolate of Cordyceps javanica (Frieder. & Bally) (Hypocreales: Cordycipitaceae) named IJ-tg19, which was isolated from soil. Spray bioassays were performed with IJ-tg19 on Myzus persicae (Sulzer) (Hemiptera: Aphididae) adults, third-instar nymphs of Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), and third-instar larvae of Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) to determine the pathogenicity of the isolate. The corrected mortality rates for all three pests were 100% when the conidia concentration was 1 × 106 conidia/ml, the lowest concentration in this study, and the median survival times (MST) were 4, 4, and 3 d. The MST shortens with increasing conidia concentration. The effects of laboratory culture conditions on the sporulation and growth of the isolate were also studied. This isolate had the greatest conidia production and fastest growth rate on malt extract agar medium at 25°C. The amount of conidia produced had positive correlation to light duration, with the highest production at 24 hr light. The growth of mycelium can adapt to a moderately alkaline environment, but the optimum conidial production occurred at the pH of 7. Our finding and research will be useful in biocontrol programs that are considering using the new isolate of C. javanica against greenhouse pests.

Chronic exposure to the star polycation (SPc) nanocarrier in the larval stage adversely impairs life history traits in Drosophila melanogaster.

Nanomaterials are widely used as pesticide adjuvants to increase pesticide efficiency and minimize environmental pollution. But it is increasingly recognized that nanocarrier is a double-edged sword, as nanoparticles are emerging as new environmental pollutants. This study aimed to determine the biotoxicity of a widely applied star polycation (SPc) nanocarrier using Drosophila melanogaster, the fruit fly, as an in vivo model. The lethal concentration 50 (LC50) value of SPc was identified as 2.14g/L toward third-instar larvae and 26.33g/L for adults. Chronic exposure to a sub lethal concentration of SPc (1g/L) in the larval stage showed long-lasting adverse effects on key life history traits. Exposure to SPc at larval stage adversely impacted the lifespan, fertility, climbing ability as well as stresses resistance of emerged adults. RNA-sequencing analysis found that SPc resulted in aberrant expression of genes involved in metabolism, innate immunity, stress response and hormone production in the larvae. Orally administrated SPc nanoparticles were mainly accumulated in intestine cells, while systemic responses were observed. These findings indicate that SPc nanoparticles are hazardous to fruit flies at multiple levels, which could help us to develop guidelines for further large-scale application.

Toxicological and molecular adverse effect of Illicium verum fruit constituents toward Bradysia procera.

Bradysia procera, a ginseng stem fungus gnat, is one of the most serious insect pests of Korean ginseng (Panax ginseng), causing significant damage to plant growth. The goal of this study was to determine the toxicity and mechanism of action of phenylpropanoids (trans-anethole and estragole) isolated from the methanol extract and hydrodistillate of Illicium verum fruit against third-instar larvae and eggs of Bradysia procera. The filter-paper mortality bioassay revealed that estragole [median lethal concentration (LC50 ) = 4.68 g/cm2 ] has a significant fumigant effect, followed by trans-anethole (LC50 = 43.92 g/cm2 ). However, estragole had the lowest toxic effect when compared to commercially available insecticides. After 7 days, estragole and trans-anethole at 75 g/cm2 inhibited egg hatchability up to 97% and 93%, respectively. At 0.09 g/cm2 , insecticides had an inhibitory effect on egg-hatching ability ranging from 88% to 94%. Furthermore, in both closed and open containers, these active constituents were able to consistently induce vapor-phased toxicity. Both estragole and trans-anethole have the ability to inhibit acetylcholinesterase (AChE), which is involved in neurotransmitter function. However, the active constituent estragole from I. verum fruit acted as a potent AChE inhibitor and had a slightly lower effect on cyclic adenosine monophosphate (AMP) than octopamine alone. This finding suggests that estragole may influence Bradysia procera neurotransmitter function via both the AChE and octopaminergic receptors. More research is needed to demonstrate the potential applications of I. verum fruit-derived products as potential larvicides and ovicides for Bradysia procera population control. © 2022 Society of Chemical Industry.

Quick shift in volatile attraction between the third and fifth instar larvae of Endoclita signifier.

Endoclita signifer is a polyphagous lepidopteran species of eucalyptus that selects its hosts in the third-instar larval period. To understand how it adapts to its host during development, we studied the olfactory responses of late-stage (fifth)-instar larvae to the dynamic chemical environment they encounter. Thirty-two volatiles from eucalyptus trunk and soil were identified, among which 14 showed electroantennal activity and five were identified as new. Further behavioral bioassay showed that both β-pinene and the imitation ratio of six key volatile in eucalyptus trunk were attractive to the fifth-instar larvae, but both eliminated and increased β-pinene in the mixture decreased the choice ratio and showed no attraction. Although E. signifer larvae shifted their attraction from o-cymene at the third-instar stage to β-pinene at the fifth-instar stage in a single volatile, the appropriate ratio of the main compounds in eucalyptus trunk volatile is the key to the behavior choice of fifth-instar larvae. The switch in olfactory attraction to different compounds between fifth- and third-instar larvae indicated an olfactory plasticity between third- and fifth-instar larvae.And the particular blend mediated the highly specialized communication interactions specificity between fifth-instar larvae and eucalyptus trunk volatile indicated the specialized host adaptation to fifth-instar larvae. This enhances understanding of how the primitive lepidopteran E. signifier, as a native pest, adapts to introduced eucalyptus. Moreover, this study provides knowledge for the screening and development of target volatiles for trapping and managing E. signifer larvae. © 2022 Society of Chemical Industry.

Bioefficacy of Euphorbia peplus latex as an antifeedant and insecticide against Gonipterus platensis larvae on Eucalyptus globulus

Background: Gonipterus platensis (Marelli) is part of the Gonipterus scutellatus species complex which consists of three species that have spread beyond their natural ranges. Due to its high reproductive potential and a capacity for intense defoliation by both larvae and adults, G. platensis causes tree growth loss and stem deformities. The antifeeding effect and insecticide efficacy of latex from petty spurge, Euphorbia peplus L. (Euphorbiaceae), on larvae of G. platensis, were evaluated through bioassays, with a view to its integrated management. Methods: Eucalyptus globulus leaves treated by immersion in Euphorbia peplus latex solutions were infested with five third-instar larvae, and the area consumed was determined at 24 h. The antifeeding effect of five latex solutions applied with a brush on Eucalyptus globulus leaves, using a random experimental design of six treatments and five replications, was also evaluated. Larval mortality was recorded daily and analysed by an ANOVA and Tukey’s test. The LC50 (lethal concentration to kill 50% of the individuals) was calculated by Probit analysis and Chi2 tests were performed. Results: The ethanolic solutions of the latex caused strong antifeeding effect, with total inhibition of larval feeding at all solutions. Larval mortality increased significantly over time up to 76%, due to the effect of the ethanol latex solutions, in all solutions by day 6. The lower LC50 values were 0.049 and 0.012% w/v on days 5 and 6, respectively. Conclusions: These results indicate that Euphorbia peplus latex is a promising bioinsecticide and a possible alternative for integrated pest management. However, further tests should be carried out.

Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle.

Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, we determined the functions of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. We uncovered that E93 was abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, our results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval-pupal-adult transition.

Transcriptome Analysis Revealed Genes Related to γ-Irradiation Induced Emergence Failure in Third-Instar Larvae of Bactrocera dorsalis.

The oriental fruit fly is a polyphagous and highly invasive economically important pest in the world. We proposed the hypothesis that radiation treatment influence RNA expression in the larvae and leads to emergence failure. Therefore, transcriptome analyses of third-instar larvae of B. dorsalis ionizing, irradiated with 60Co-γ at 116Gy, were conducted and compared with the controls; a total of 608 DEGs were identified, including 348 up-regulated genes and 260 down-regulated ones. In addition, 130 SNPs in 125 unigenes were identified. For the DEGs, the most significantly enriched GO item was hemolymph coagulation, and some of the enriched pathways were involved in digestive processes. The subsequent validation experiment confirmed the differential expression of six genes, including sqd, ENPEP, Jhe, mth, Notch, and Ugt. Additionally, the 3401:G->A SNP in the Notch gene was also successfully validated. According to previous research, this was the first comparative transcriptome study to discover the candidate genes involved in insect molt to pupae. These results not only deepen our understanding of the emerging mechanism of B. dorsalis but also provide new insights into the research of biomarkers for quarantine insect treatment with the appropriate dose of radiation.

Cyclosporin A as a Potential Insecticide to Control the Asian Corn Borer Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae).

Simple SummaryWhile chemical insecticides are conventionally effective for pest control, they also cause severe problems, such as insect resistance and environmental pollution. Alternative insecticides with distinct mechanisms are needed. Cyclosporin A (CsA), a polypeptide produced by fungi, is widely used as an immunosuppressant in organ transplantation. This study used Ostrinia furnacalis, a major pest of maize in Asia, as a model to investigate the insecticidal activity of CsA. CsA had marked insecticide activity against O. furnacalis larvae. Furthermore, sublethal doses of CsA caused negative effects on larval, pupal and adult development. CsA treatments suppressed reproduction, significantly decreasing the mating rate, ovary size, and numbers and hatchability of eggs. CsA showed excellent combined toxicities when mixed with five common insecticides, respectively. Importantly, CsA acts by suppressing calcineurin, which represents a novel insecticidal target. Thus, CsA has potential as a new insecticide for sustainable pest management.The long-term use of chemical insecticides has caused serious problems of insect resistance and environmental pollution; new insecticides are needed to solve this problem. Cyclosporin A (CsA) is a polypeptide produced by many fungi, which is used to prevent or treat immune rejection during organ transplantation. However, little is known about the utility of CsA as an insecticide. Therefore, this study evaluated the insecticidal activity of CsA using Ostrinia furnacalis as a model. The results demonstrated that CsA was toxic to O. furnacalis with LC50 values of 113.02 μg/g and 198.70 μg/g for newly hatched neonates and newly molted third-instar larvae, respectively. Furthermore, CsA treatment had sublethal effects on the development of O. furnacalis, and significantly reduced the fecundity of adults; this suggests that CsA has great potential to suppress O. furnacalis populations. Further analysis revealed that CsA suppressed calcineurin activity in larvae. CsA had independent or synergistic toxic effects on O. furnacalis when combined with β-cypermethrin, indoxacarb, emamectin benzoate, azadirachtin, and the Bacillus thuringiensis toxin Cry1Ac, which suggests that CsA can help prevent or manage resistance. Our study provides detailed information on the potential of CsA as an insecticide for controlling lepidopterans.

Investigation of Indigenous Entomopathogenic Nematodes in Guangxi and Its Biological Control of Spodoptera frugiperda

Spodoptera frugiperda has caused serious economic damage to various crops. Entomopathogenic nematodes (EPNs) can be used as biological control agents for many pests, including lepidopteran insects. In this study, 218 soil samples were collected from 46 sites in Guangxi, and EPNs were detected in 15 samples. The ITS region of the rDNA gene was used for the molecular identification of isolated nematodes. In total, four and eleven identified populations belonged to Heterorhabditis and Oscheius, respectively. A series of bioassays were conducted to examine the virulence of EPN isolates from Guangxi to control the larvae and pupae of S. frugiperda. The mortality of the third-instar larvae caused by EPNs was concentration dependent. The same dose of EPNs was used to control the third and sixth-instar larvae of S. frugiperda, and the virulence was lower in the sixth-instar larvae. S. frugiperda pupae were treated with different EPNs strains, and the adult eclosion rate of the treated group was significantly lower than that of the sterile water control group (93.3%). Therefore, EPNs could significantly inhibit the eclosion of S. frugiperda pupae. This study provides important information for the biological control of S. frugiperda with EPNs.

The transcriptomic signature of responses to larval crowding in Drosophila melanogaster.

Intraspecific competition at the larval stage is an important ecological factor affecting life-history, adaptation and evolutionary trajectory in holometabolous insects. However, the molecular pathways underpinning these ecological processes are poorly characterized. We reared Drosophila melanogaster at three egg densities (5, 60, and 300 eggs/mL) and sequenced the transcriptomes of pooled third-instar larvae. We also examined emergence time, egg-to-adult viability, adult mass, and adult sex-ratio at each density. Medium crowding had minor detrimental effects on adult phenotypes compared to low density and yielded 24 differentially expressed genes (DEGs), including several chitinase enzymes. In contrast, high crowding had substantial detrimental effects on adult phenotypes and yielded 2107 DEGs. Among these, upregulated gene sets were enriched in sugar, steroid and amino acid metabolism as well as DNA replication pathways, whereas downregulated gene sets were enriched in ABC transporters, taurine, Toll/Imd signaling, and P450 xenobiotics metabolism pathways. Overall, our findings show that larval crowding has a large consistent effect on several molecular pathways (i.e., core responses) with few pathways displaying density-specific regulation (i.e., idiosyncratic responses). This provides important insights into how holometabolous insects respond to intraspecific competition during development.

Functional validation of CYP304A1 associated with haedoxan A detoxification in Aedes albopictus by RNAi and transgenic drosophila.

Insect cytochrome P450 monooxygenases play important roles in the detoxification metabolism of endogenous and exogenous compounds. Haedoxan A (HA) from Phryma leptostachya L. is a highly efficient natural pesticide used to control houseflies and mosquitos. CYP4C21 and CYP304A1 were previously demonstrated to be transcriptionally increased in Aedes albopictus in response to HA exposure, but their involvement in HA metabolism is unknown. Our data showed that CYP304A1 expression levels in A. albopictus were highest in third-instar larvae, and the expression level of CYP4C21 decreased significantly with the growth of instars, with the lowest occurring in the pupal stage. Compared with the control, the silencing of CYP304A1 and CYP4C21 genes by chitosan nanoparticle-mediated RNA interference could deplete 58.2% and 54.0% of the expression of corresponding genes, respectively. The bioassay data showed that knocking down the expression of CYP304A1 increased the mortality of A. albopictus when exposed to HA at LC30 and LC50 doses, but did not significantly increase mortality after silencing CYP4C21. Our data demonstrated that CYP304A1, but not CYP4C21, may be involved in HA detoxification. Moreover, the resistance ratio of CYP304A1 overexpressing flies was approximately 2-fold higher than that of the control line. The metabolized product of HA by CYP304A1 needs to be further confirmed by in vitro expression. This finding showed that inducibility was not always linked to detoxifying capabilities, and enhanced our understanding of the molecular basis of HA metabolic detoxification in A. albopictus. © 2022 Society of Chemical Industry.

Mass Purification Protocol for Drosophila melanogaster Wing Imaginal Discs: An Alternative to Dissection to Obtain Large Numbers of Disc Cells.

Simple SummaryDrosophila melanogaster, also known as the fruit fly, is a widely used organism model, especially for genetic studies or as a model for pathologies. The Drosophila genome is well known and conserved within humans, thus allowing biologists to obtain numerous mutants and transgenic flies from the fruit flies. Gene function studies at the cellular and molecular levels are often performed using extracts of larval tissues. Due to their small size, it is difficult to dissect substantial amounts of these tissues for performing genomic or proteomic experiments. This paper develops a simple method to purify larval tissues en masse. This protocol preserves tissue integrity in the same way as manual dissection; the protocol is achievable by individual researchers and allows the purification of different samples simultaneously.Drosophila melanogaster imaginal discs are larval internal structures that become the external organs of the adult. They have been used to study numerous developmental processes for more than fifty years. Dissecting these imaginal discs for collection is challenging, as the size of third-instar larvae organs is typically less than 1 mm. Certain experimental applications of the organs require many cells, which requires researchers to spend several hours dissecting them. This paper proposes an alternative to dissection in the form of a mass enrichment protocol. The protocol enables the recovery of many wing imaginal discs by grinding large quantities of third-instar larvae and separating the organs using filtration and a density gradient. The wing imaginal discs collected with this protocol in less than three hours are as well preserved as those collected by dissection. The dissociation and filtration of the extract allow the isolation of a large amount of wing imaginal disc cells.

Closing the Brief Case: Furuncular Myiasis Caused by the Third-Instar Larva of a Bot Fly in the Genus Cuterebra.

Closing the Brief Case: Furuncular Myiasis Caused by the Third-Instar Larva of a Bot Fly in the Genus Cuterebra.