4th Instar Larvae Research Articles

Bio-control potential of Bacillus thuringiensis (Bacillales: Bacillaceae) and Metarhizium anisopliae (Hypocreales: Clavicipitaceae) against Chilo partellus Swinhoe (Lepidoptera: Pyralidae)

The efficacy of most synthetic insecticides has been compromised against Chilo partellus (Swinhoe) due to its cryptic feeding behavior, hence there is a dire need to find other eco-friendly effective management approaches to combat this notorious pest. The susceptibility of 2nd and 4th larval instars of C. partellus was assessed against Bacillus thuringiensis var. kurstaki (Bt-k) (Berliner) and Metarhizium anisopliae (Mechnikov) under controlled conditions. The larvae were treated with individual or combined treatments of a single dose rate of B. thuringiensis (Bt: 0.75 µg/g) and three conidial concentrations (1x104, 1x106 and 1x108 conidia/ml) of M. anisopliae. The individual and combined application of both entomopathogens significantly affected the mortality, pupation, adult emergence, mycosis and sporulation of both larval instars of C. partellus. Maximum larval mortality (2nd instar: 94.63% and 4th instar: 91.43%) was recorded in combined applications of M. anisopliae (1x108 conidia/ml) and B. thuringiensis (0.75 µg/g). The same integrated treatment also caused significant declines in pupation (2nd: 0.00% and 4th: 6.66%) and adult emergence (2nd: 0.00% and 4th: 0.00%) of C. partellus as compared to rest of the experimental treatments. Moreover, a significantly higher rate of mycosis and sporulation were recorded in both larval instars (2nd: 88.52% and 143.96 conidia/ml) and (4th: 82.04% and 131.85 conidia/ml), when exposed to lower individual concentration of M. anisopliae (1×104 conidia/ml). The current results suggest that integrated application of M. anisopliae and B. thuringiensis could be a sustainable management tool for C. partellus in maize crop.

Evaluation of entomopathogenic nematode (EPN) isolate, Heterorhabditis indica of Vidarbha region, against the tobacco cutworm, Spodoptera litura

The present study on pathogenicity of entomopathogenic nematodes against Spodoptera litura in laboratory conditions was undertaken during 2020-21, with the aim to ascertain the effectiveness of entomopathogenic nematodes, against an obnoxious cosmopolitan pest S. litura. Experiments were conducted by using entomopathogenic nematode (EPN) isolate Heterorhabditis indica (CICR-Guava), on filter paper, against Galleria mellonella and S. litura at the treatment dose of 10, 20, 30, 40, 60, 80 and 100 IJs/100µl along with control (Sterile distilled water). The results of our study revealed that, EPN isolate H. indica (CICR-Guava) caused 100% mortality at the treatment dose of 40 IJs/100µl within 72 h of infection in 5th instar larvae of G. mellonella and in case of S. litura, 100% mortality was recorded within 72 h of infection at the treatment dose of 100 IJs/100µl in 3rd instar larvae, which was found more susceptible. The median lethal concentration of H. indica (CICR-Guava) for 5th instar larvae was 2.29 IJs/100µl. The result of reproductive potential of isolates of entomopathogenic nematodes revealed that the highest yield was obtained from 5th instar larvae of G. mellonella at treatment dose of 100 IJs/100µl 278667 IJs per larva. In case of S. litura, the highest yield obtained was 152533 IJs. It could be concluded that, there was a positive correlation between nematode treatment concentration, time of exposure and the insect mortality of the tobacco cut worm and multiplication rate of IJs increased with increase of exposure time and size of larvae. This EPN isolate, H. indica (CICR-Guava) can be suggested as biocontrol agents for the control of S. litura in the Vidarbha region.

MOSQUITO LARVICIDAL POTENCY OF SELECTED HALOPHYTE SPECIES AND THEIR MODULATION ON ACETYLCHOLINESTERASE AND GLUTATHIONE S-TRANSFERASE AGAINST DENGUE VECTOR: Aedes aegypti

A mangrove is an ecosystem that represents a natural product reservoir of biotechnological interest. They have been advocated as environmentally benign alternatives to synthetic pesticides against all vector mosquitos. The purpose of this study was to determine the larvicidal activities of selected mangrove plant extracts against Aedes aegypti mosquito larvae. Methanol extracts of three selected halophytes viz. Lumnitzera racemosa, Bruguiera cylindrica, and Excoecaria agallocha were made, phytochemically investigated and evaluated against A. aegypti early 4th instar larvae. Acetylcholinesterase (AChE) activity and Glutathione S-transferase (GST) activity were evaluated, followed by DNA extraction and fragmentation tests. GC-MS technique was used to determine the presence of bioactive chemicals. Alkaloids, flavonoids, terpenoids, anthocyanin, coumarins, phenolic compounds, and quinones were found in all plants. The best larvicidal activity was recorded in the L. racemosa flower buds (LC50- 0.766 mg/ml) and E. agallocha stem extract (LC50- 0.986 mg/ml). L. racemosa flower buds and E. agallocha stem exhibited higher inhibition in Acetylcholinesterase (AChE) activity with the rate of 1.39 μmol ACT/min/mg protein and 1.65 μmol ACT/min/mg protein (p < 0.05) in treated larvae. In the case of L. racemosa flower buds, however, enhanced GST activity was detected. Plant extracts were found not to cause any fragmentation activity in the larval DNA. 5 major peaks of chemical compounds were analyzed in L. racemosa and E. agallocha. Because of the possible larvicidal action of selected halophyte extracts, they can be utilized as ecologically friendly mosquitocide for eradicating dengue vector larvae in their early stages.

The TGF-β Receptor Gene Saxophone Influences Larval-Pupal-Adult Development in Tribolium castaneum.

The transforming growth factor-β (TGF-β) superfamily encodes a large group of proteins, including TGF-β isoforms, bone morphogenetic proteins and activins that act through conserved cell-surface receptors and signaling co-receptors. TGF-β signaling in insects controls physiological events, including growth, development, diapause, caste determination and metamorphosis. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of the type I TGF-β receptor, saxophone (Sax), in mediating development. Developmental and tissue-specific expression profiles indicated Sax is constitutively expressed during development with lower expression in 19- and 20-day (6th instar) larvae. RNAi knockdown of Sax in 19-day larvae prolonged developmental duration from larvae to pupae and significantly decreased pupation and adult eclosion in a dose-dependent manner. At 50 ng dsSax/larva, Sax knockdown led to an 84.4% pupation rate and 46.3% adult emergence rate. At 100 ng and 200 ng dsSax/larva, pupation was down to 75.6% and 50%, respectively, with 0% adult emergence following treatments with both doses. These phenotypes were similar to those following knockdowns of 20-hydroxyecdysone (20E) receptor genes, ecdysone receptor (EcR) or ultraspiracle protein (USP). Expression of 20E biosynthesis genes disembodied and spookier, 20E receptor genes EcR and USP, and 20E downstream genes BrC and E75, were suppressed after the Sax knockdown. Topical application of 20E on larvae treated with dsSax partially rescued the dsSax-driven defects. We can infer that the TGF-β receptor gene Sax influences larval-pupal-adult development via 20E signaling in T. castaneum.

Insecticidal activities of the local entomopathogenic nematodes and cell-free supernatants from their symbiotic bacteria against the larvae of fall webworm, Hyphantriacunea

The fall webworm (FWW), Hyphantria cunea Drury (Lepidoptera: Erebidae), is an invasive and polyphagous insect pest of many economically important crops such as hazelnuts, apple, and mulberry. Recently, there have been an increasing number of reports about the damaging activities of FWW from hazelnut growing areas of Turkey indicating that currently existing control methods fail to satisfy the expectations of growers. Entomopathogenic nematodes (EPNs) in the Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) families and the symbiotic bacteria they carry in their intestine have a great potential for the management of many agriculturally important pests. In this study, the symbiotic bacteria of local EPN species (Heterorhabditis bacteriophora AVB-15, Steinernema feltiae KCS-4S, and Steinernema bicornotum MGZ-4S) recovered from the central Anatolia region was characterized using recA gene region as Photorhabdus luminescens, Xenorhabdus bovienii and Xenorhabdus budapestensis. The contact (25, 50, 100, 200 IJs/Petri) and oral efficacies of the infective juveniles (IJs) (25, 50, 100, 200 IJs/leaf) of these EPN isolates determined on 3rd/4th instar larvae, and cell-free supernatants from the identified symbiotic bacteria were evaluated separately on the 3rd and 4th larval instars of FWW in Petri dish environment under laboratory conditions (25 ± 1 °C, 60% of RH). In the Petri dish bioassays of EPN species, the most pathogenic isolate at the 1st DAT and 4th DAT was S. feltiae which caused 50% mortality at the highest concentration (200 IJs/Petri) and the highest mortality rate (97.5%) were achieved at 4th DAT by H. bacteriophora AVB-15 isolate. Surprisingly, the mortality rates were generally higher at the lowest concentrations and 82.5% mortality were reached 4th DAT by S. bicornotum at the lowest concentration (25 IJs/leaf) in the leaf bioassays. Mortality rates were higher in both Petri dish and filter paper efficacies of cell-free supernatants at the 2nd DAT and the highest mortality (87.5%) was reached in the contact efficacy studies when applied X. bovienii KCS-4S strain. The results suggest that the tested EPN species and CFSs have good potential for biological control of the larvae of FWW and can contribute to the IPM programs of FWW. However, the efficacy of both IJs of EPNs and CFSs of their symbiotic bacteria on larvae of FWW requires further studies to verify their efficiency in the field.

Biocidal potential of indigenous isolates of Entomopathogenic Nematodes (EPNs) against tobacco cutworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

BackgroundEntomopathogenic nematodes (EPNs) have been regarded as the most convenient strategy for insect pest management. The native strains of EPNs: Heterorhabditis bacteriophora EUPT-SD, H. bacteriophora EUPT-R, H. bacteriophora EUPT-KN, H. bacteriophora EUPT-K and H. bacteriophora EUPT-H isolated from mid-Himalayan region of Himachal Pradesh were tested in laboratory for their multiplication and virulence against 3rd and 4th larval instars of the tobacco cutworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae), the serious polyphagous pest affecting a wide range of agricultural crops worldwide.ResultsAll the EPN strains were effective against 3rd and 4th larval instars of S. litura. Insect mortality reached 90–96% after 96 h at nematode concentrations of 150 infective juveniles (IJs)/ml. The insect mortality was also recorded at low concentrations of IJs, but the most exposure period was required. High virulence was shown by H. bacteriophora EUPT-SD 96 and 94%, followed by H. bacteriophora EUPT-R 92 and 90%, H. bacteriophora EUPT-KN 92 and 90%, H. bacteriophora EUPT-K 92 and 90% and H. bacteriophora EUPT-H 92 and 90%, respectively, against 3rd and 4th larval instars in terms of reproductive potential and killing. All the insects were alive in the absolute control.ConclusionUtilization of EPNs for the management of S. litura may be the best method to overcome the insect resistance problems and to manage the population of this insect pest. It may be an effective method and may be a partial substitute of synthetic insecticides, thus minimizing the excessive use of synthetic chemicals. The results demonstrated the potential of indigenous EPNs isolates against S. litura, but before further recommendation, multiplication field trials need to be conducted to confirm their efficacy at farm level.

Cladophialophora bantiana metabolites are efficient in the larvicidal and ovicidal control of Aedes aegypti, and Culex quinquefasciatus and have low toxicity in zebrafish embryo

Mosquitoes' current insecticide resistance status in available public health insecticides is a serious threat to mosquito control initiatives. Microbe-based control agents provide an alternative to conventional pesticides and insecticides, as they can be more targeted than synthetic insecticides. The present study was focused on identifying and investigating the mosquitocidal potential of Cladophialophora bantiana, an endophytic fungus isolated from Opuntia ficus-indica. The Cladophialophora species was identified through phylogenetic analysis of the rDNA sequence. The isolated fungus was first evaluated for its potential to produce metabolites against Aedes aegpti and Culex quinquefasciatus larvae in the 1-4th instar. The secondary metabolites of mycelium extract were assessed at various test doses (100, 200, 300, 400, and 500 μg/mL) in independent bioassays for each instar of selected mosquito larvae. After 48 h of exposure, A. aegypti expressed LC50 values of 13.069, 18.085, 9.554, and 11.717 μg/mL and LC90 = 25.702, 30.860, 17.275, and 19.601 μg/mL; followed by C. quinquefasciatus LC50 = 14.467, 11.766, 5.934, and 7.589 μg/mL, and LC90 = 29.529, 20.767, 11.192, and 13.296 μg/mL. The mean % of ovicidal bioassay was recorded 120 h after exposure. The hatchability (%) was proportional to mycelia metabolite concentration. The enzymatic level of acetylcholinesterase in fungal mycelial metabolite treated 4th instar larvae indicated a dose-dependent pattern. The GC–MS profile of C. bantiana extracts identified five of the most abundant compounds, namely cyclobutane, trans-3-undecene-1,5-diyne, 1-bromo-2-chloro, propane, 1,2,3-trichloro-2-methyl-, 5,5,10,10-tetrachlorotricyclo, and phenol, which had the killing effect in mosquitoes. Furthermore, the C. bantiana fungus ethyl acetate extracts had a strong larvicidal action on A. aegypti and C. quinquefasciatus. Finally, the toxicity test on zebrafish embryos revealed the induction of malformations only at concentrations above 1 mg/mL. Therefore, our study pioneered evidence that C. bantiana fungal metabolites effectively control A. aegypti and C. qunquefasciastus and show less lethality in zebrafish embryos at concentrations up to 500 μg/mL.

Green silver nanoparticles against Helicoverpa armigera and its effects on biochemical, morphological and histological aspects

The aim of this study was to evaluate the effect of green synthesized silver nanoparticles using aqueous leaf extract of Ricinus communis (castor plant) on the life cycle Helicoverpa armigera. The synthesized AgNPs were identified using different microscopic techniques: UV-Visible spectroscopy, Scanning electron microscopy (SEM), Energy-disruptive X-ray spectroscopy (EDX) and Transmission electron microscopy (TEM). Insecticidal activity of AgNPs was studied using leaf dipping method. The biochemical studies were analyzed at LC50 of AgNPs using different methods (total carbohydrate using phenol-sulphuric acid reaction of Dubois method, total protein using Bradford method, total lipid using sulfo-phospho vanillin of Knight method and non-specific esterases using Van-Asperan method, respectively). UV-vis spectrophotometer confirmed the spectra at 445 nm of AgNPs. Spherical shaped AgNPs of 8.96 nm size were confirmed by TEM and SEM. EDX spectra confirm the chemical composition AgNPs. Higher mortality was observed in 6th instar larvae (70-80% at LC50 175 ppm) than the other instars. The pupa and adult of H. armigera also exhibited toxicity against the AgNPs. Effect of AgNPs treatment was investigated on the midgut of 5th instar larva of H. armigera using the transmission electron microscope. Accumulation of AgNPs in cell organelles was confirmed by ultra structural studies of insect midgut cell using TEM. The biochemical components of 5th instar larvae showed that, the amount of total carbohydrate, total protein, total lipid and non-specific esterases were significantly decreased After 24 h treatment at LC50 of AgNPs. Now, we can summarise that, synthesis of AgNPs using R. communis is a green route to control pest population. The synthesized nanoparticles could be used for the improvement of new botanical nano-insecticide or nano-formulation after successful field trials.

Larvicidal and pupicidal activity of crude ethyl acetate extract fraction-7a of Cymodocea serrulata on Culex quinquefasciatus

Finding a potential bioactive compound against a specific mosquito vector is an urgent requirement. Thus, this research was designed to separate the significant compounds present in the crude ethyl acetate extract of Cymodocea serrulata through Thin Layer Chromatography (TLC), Column Chromatography (CC), and High Performance Liquid Chromatography (HPLC). Furthermore, the larvicidal and pupicidal activity of different concentrations (100, 300, 500, 700, and 900 g mL−1) of separated fraction against 4th instar larvae of Culex quinquefasciatus in 24 h of treatment. Interestingly, 7 fractions were obtained from crude ethyl acetate extract by TLC. One fraction (7a) from out of 7 was further separated into fractions 7a and 7b through CC and preparative TLC. Furthermore, the HPLC analysis result revealed that the fraction-7a contained 2 major peaks and 25 minor peaks. The fraction 7a was selected based on the Rf value and examined their larvicidal and pupicidal activity on 4th instar larvae and pupa of C. quinquefasciatus. Interestingly, the increased concentration (900 µg mL−1) of fraction-7a of ethyl acetate extracts of C. serrulata showed excellent larvicidal and pupicidal activities as 98.66 ± 1.88 % and 98 ± 5.65 %, respectively. These findings imply that the fraction 7a of C. serrulata ethyl acetate extract could be used as a mosquito vector control agent.

Parasitic behavior of Pyemotes zhonghuajia (Trombidiformes: Pyemotidae) on fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

Fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a destructive, migratory pest that seriously damages corn, sorghum, and other crops. The “insect killer” Pyemotes zhonghuajia (Trombidiformes: Pyemotidae) is an ectoparasitic mite of agricultural and forestry pests in China. To explore the biological control efficiency of P. zhonghuajia on S. frugiperda, we evaluated the parasitic selectivity of P. zhonghuajia on S. frugiperda of different life stages, and distribution of P. zhonghuajia on different body parts of S. frugiperda larvae and pupae, food intake of S. frugiperda larvae affected by P. zhonghuajia density, and field population dynamics of S. frugiperda in relation to the releasing population density of P. zhonghuajia. We found that P. zhonghuajia preferred 4th- to sixth-instar larvae and pupae of S. frugiperda for parasitism, and the ventral part of abdominal segments of fourth-instar larvae and the ventral part of pupal thorax for feeding. Freezing of S. frugiperda 4th instar larvae and pupae had little effect on the distribution of P. zhonghuajia on the surface of these two host stages. The parasitism of P. zhonghuajia regardless of mite population size (i.e., 5, 10, or 20 mites) could significantly decrease the cumulative food intake of fifth-instar larvae of S. frugiperda. Releasing two and three boxes (50,000 mites/box) of P. zhonghuajia significantly suppressed the field population of S. frugiperda on sorghum plants/25 m2 within 7 days. Our results show that P. zhonghuajia may be a potential biological control agent and can be used for biological control of S. frugiperda in the field.

Near Infrared Spectroscopy as a Traceability Tool to Monitor Black Soldier Fly Larvae (Hermetia illucens) Intended as Animal Feed

The demand for animal proteins, especially from pork and poultry, is projected to increase significantly due to rapid growth in population and underlying socio-economic conditions. Livestock rearing using conventional feed ingredients is becoming challenging due to climate change and several other factors, thereby suggesting the need for alternative, viable and sustainable animal feed sources. The use of black soldier fly larvae (BSFL) (Hermetia illucens) as a component in animal feed is a promising candidate due to their ability to valorise different organic waste streams. The nutrient composition of BSFL reared on organic waste streams is also comparable to that of several conventional animal feed ingredients and varies depending upon the feed, rearing conditions, and the morphological stage of the larvae. The identification of organic waste is of importance as it can determine not only the composition but also the safety issues of BSFL as an animal feed ingredient. The objective of this study was to determine the ability of near-infrared (NIR) spectroscopy to trace the food waste used to grow BSFL. Samples of BSFL (5th and 6th instar BSFL; n = 50) obtained from a commercial production facility were analysed using NIR spectroscopy. Partial least squares discriminant analysis (PLS-DA) was employed to develop the models. The outcomes of this study revealed that NIR spectroscopy could distinguish different larval instars and suggested the importance of larval instars in developing calibration models for traceability applications. The developed PLS-DA model could predict the feed source used for rearing the 5th instar larvae (R2 value: 0.89) and 6th instar pre-pupae (R2 value: 0.91). This suggests that NIR spectroscopy could be used as a non-invasive traceability tool for BSFL and to assist in selecting the suitable time frame for larvae harvesting in commercial facilities.

Evaluation of Entomopathogenic Nematodes against Red Palm Weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae).

Simple SummaryThe red palm weevil is considered the most notorious pest for different species of palms around the globe. Due to the concealed nature of the red palm weevil, the use of biocontrol agents, especially EPNs, is considered most effective. Among biocontrol agents, entomopathogenic nematodes provide effective control of the different developmental stages of the red palm weevil. In the current investigation, the infective capabilities of four different species of entomopathogenic nematodes were investigated against larvae (5th and 6th instars), pupae enclosed in their cocoons, and adult red palm weevil (newly formed), under laboratory and field conditions. Our results indicated that the S. carpocapsae and H. bacteriophora were the most effective EPN species against different developmental stages of the red palm weevil under laboratory as well as field conditions. The larval stage of the red palm weevil was found to be the most susceptible to infection by EPNs compared with pupal and adult stages. From the present findings, we can infer that S. carpocapsae and H. bacteriophora are the most effective EPN strains and recommend their use in the sustainable management of the red palm weevil.Entomopathogenic nematodes play a pivotal role as biocontrol agents for different species of insect pests, including the red palm weevil. In the current investigation, the infective capabilities of four species of entomopathogenic nematodes, including Hetrerorhabditis bacteriophora, Steinernema feltiae, Steinernema glaseri, and Steinernema carpocapsae, were evaluated against larvae, pupae, and adult red palm weevil under laboratory and field conditions. The pathogenic potential of selected nematode species was assessed based on dissection and adult emergence of weevils. Our results indicated that S. carpocapsae and H. bacteriophora, with a respective 94.68 and 92.68% infection rate, were the most effective EPN species against red palm weevil larvae. Focusing on adult emergence, the aforementioned EPNs were comparatively less pathogenic and resulted in 63.60 and 60.20% infested pupae, respectively. It is noted that adult emergence is the better option to evaluate the pathogenic potential of EPNs, compared with the dissection of insects. The S. carpocapsae was found to be most effective against the 6th instar larvae of the red palm weevil and caused 100% mortality at 240 h after treatment. On the other hand, S. glaseri and S. feltiae were found to be the least pathogenic and caused 70 and 76% mortality, respectively. All of the evaluated nematode species were found to be highly infective under field conditions. The S. carpocapsae was found to be the most pathogenic, causing 83.60% mortality of the red palm weevil. However, the tested nematodes were found most effective against larvae, followed by adult weevils, but their effect was minimal against the pupae of red palm weevils. Based on these findings, we conclude that the S. carpocapsae and H. bacteriophora could be used as a sustainable option for the efficient management of the red palm weevil.

Dynamics of the intestinal bacterial community in black soldier fly larval guts and its influence on insect growth and development.

Black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is a prominent insect for the bioconversion of various organic wastes. As a saprotrophic insect, the BSF inhabits microbe-rich environments. However, the influences of the intestinal microorganisms on BSF growth and development are not very clear. In this study, the dynamics of the intestinal bacterial community of BSF larvae (BSFL) were analyzed using pyrosequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most prevalent bacterial phyla in the intestines of all larval instars. The dynamic changes in bacterial community compositions among different larval instars were striking at the genus level. Klebsiella, Clostridium, Providencia, and Dysgonomonas were the relatively most abundant bacteria in the 1st- to 4th-instar BSFL, respectively. Dysgonomonas and Providencia also dominated the 5th- and 6th-instar larvae, at ratios of 31.1% and 47.2%, respectively. In total, 148 bacterial strains affiliated with 20 genera were isolated on different media under aerobic and anaerobic conditions. Among them, 6 bacteria, BSF1-BSF6, were selected for further study. The inoculation of the 6 isolates independently into germ-free BSFL feeding on an artificial diet showed that all the bacteria, except BSF4, significantly promoted BSF growth and development compared with the germ-free control. Citrobacter, Dysgonomonas, Klebsiella, Ochrobactrum, and Providencia promoted BSF development significantly by increasing the weight gains of larvae and pupae, as well as increasing the prepupae and eclosion rates. In addition, Citrobacter, Klebsiella and Providencia shortened the BSF life cycle significantly. The results illustrate the promotive effects of intestinal bacteria on BSF growth and development.

Effect of Hg stress in Larix olgensis on the growth, antioxidant and detoxifying enzymes of the Lymantria dispar

To understand the physiological response mechanisms of Lymantria dispar larvae to heavy metal stress, their host, 2-year-old Larix olgensis seedlings grown in pots, were treated with three different concentrations of HgCl2 (Hg). Hg accumulation and tannin content in larch needles were measured. The growth and detoxification ability of the larvae were analyzed after being fed on the Hg-stressed needles. The results showed that Hg concentration in larch needles increased with Hg treatment concentration increase, while the tannin content decreased. The larval body mass was significantly decreased. The responses of the larval antioxidant and detoxifying enzymes to Hg stress appeared to differ, with the superoxide dismutase (SOD) and peroxidase (POD) activities in 4th instar larvae were significantly higher than CK. In contrast, the acid phosphatase (ACP), alkaline phosphatase (AKP) and glutathione S-transferase (GST) activities in 4th instar larvae were significantly lower than CK. This result indicated that Hg could inhibit the growth of larvae along the food chain. The larval antioxidant and detoxification enzymes produced adaptive responses to the Hg stress, which might partially alleviate the oxidative damages caused by Hg.

The parasitic impact of Romanomermis iyengari Welch (Nematoda: Mermithidae) on the survival and biology of the common mosquito, Culex pipiens L. (Diptera: Culicidae)

The extensive use of chemical insecticides to control mosquitoes led to the development of insecticide resistance and environmental health hazards. This caused a surge in interest in eco-friendly biocontrol agents. The present study aimed to explore the susceptibility of different instar larvae of the common mosquito, Culex pipiens Linnaeus, 1758, to the mermithid nematode, Romanomermis iyengari Welch, 1964. Moreover, the effects of R. iyengari on the mosquito pupal developmental time, adult emergence, longevity, female fecundity, as well as egg-hatching rate were determined after larval treatment with an LC50 quantity of the nematode pre-parasites. Different instars of Cx. pipiens (1st–4th) were exposed separately to R. iyengari at concentrations of 1–6 pre-parasites/mosquito larva. Mortality rates of mosquito larvae were observed and the LC50 values were calculated. The estimated LC50 values for the 1st–4th larval instars were 3.18, 2.73, 3.79 and 4 pre-parasites/larva, respectively. Mean percent mortality of the 1st–4th larval instars ranged from 10–94%, 16–100%, 4–100% and 0–52%, respectively. The results indicated that exposure of 4th larval instar to the LC50 of R. iyengari pre-parasites significantly prolonged the duration of pupal development, reduced the percentage of emerged adults and reduced mosquito female fecundity compared with the control. In contrast, adult longevity and the egg-hatching rate did not differ between the control and the exposed group. In conclusion, this isolate of R. iyengari could be a promising biocontrol agent for Egyptian Cx. pipiens. Other trials are required to assess the biocontrol potential of this parasite in field conditions.

Diversity of fungi associated with Monochamusalternatus larval habitats in Bursaphelenchusxylophilus-infected Pinusmassoniana and identification of two new ophiostomatalean species (Ascomycota, Ophiostomatales).

Bursaphelenchusxylophilus, a pathogenic pine wood nematode (PWN), is responsible for pine wilt disease (PWD), which has caused significant economic and ecological damage worldwide, particularly in East Asia. Multiple biological factors, such as the beetle vector Monochamus, symbiotic bacteria and associated fungi, are involved in the disease infection cycle. This study isolated and identified the fungal communities of Monochamusalternatus larval galleries and pupal chambers from different instars through field investigation, morphological observation and multi-locus DNA sequence analyses in Zhejiang Province, China. A total of 255 and 454 fungal strains were isolated from M.alternatus galleries and pupal chambers infected with PWN, from the 2nd-3rd and 4th-5th instar larvae, respectively. A total of 18 species of fungi were identified, 14 species were isolated from the 2nd-3rd instar larval galleries and six species from the galleries and pupal chambers of the 4th-5th instar larvae. Amongst them were six species belonging to four genera of ophiostomatalean fungi, including two novel species, Graphilbumxianjuensis sp. nov. and Ophiostomataizhouense sp. nov. and four known species, Ceratocystiopsisweihaiensis, Ophiostomaips, Sporothrixzhejiangensis and S.macroconidia. The findings revealed that the fungal diversity and abundance of the 2nd-3rd instar larvae differed markedly from those of the 4th-5th instar larvae. This difference could be the result of fungal succession. This study provides a thorough understanding of the fungi associated with PWD and lays the groundwork for future research.

Genome-wide survey of open chromatin regions in two swallowtail butterflies Papilio machaonand P. bianor.

Papilio machaon was assigned as the type species for all butterflies by Linnaeus and P. bianor is a congener but exhibits a great difference in morphology (especially larva and adult color pattern) and larval host plants from P. machaon. Thus, they are the ideal models to investigate genetic mechanisms underlying morphology and plasticity between congeners. The reference genomes of both species were dissected in our previous studies, but little is known about their regulatory genome and the epigenetic regulation of gene expression throughout developmental stages. Here, we profiled the chromatin accessibility and gene expression of three developmental stages (the 4th instar larva [L4], the 5th instar larva [L5], and pupa [P]) using transposase accessible chromatin sequencing (ATAC-seq) and RNA-seq. Results showed that many accessible chromatin peakswere identified at three developmental stages (peak number, P. machaon: 44,977 [L4], 36,919 [L5], 47,147 [P]; P. bianor: 20,341 [L4], 44,668 [L5], 62,249 [P]). Moreover, the number of differentially accessiblepeaks and differentially expressed genesbetween larval stages of each butterfly species are significantly fewer than that between larval and pupal stages, suggesting a higher similarity within larvae and a significant difference between larvae and pupae. This study added the annotated information of chromatin accessibility genome-wide of the two papilionid species and will promote the investigation of gene regulation in butterfly evolution.

Lithium chloride leads to concentration dependent brood damages in honey bee hives (Apis mellifera) during control of the mite Varroa destructor

Lithium chloride (LiCl) has a high efficacy against Varroa destructor and a good tolerability for adult bees but the effect of LiCl on the honey bee brood has not been taken into consideration yet. We quantified the mortality of larvae fed with different concentrations of LiCl. For artificially reared larvae already, a concentration of 1 mM had significant toxic effects while under colony conditions, 10 mM was well tolerated. However, a chronic application of the effective concentration of 25 mM elicited brood mortalities between 60 and 90%. Shorter feeding periods of 2 or 4 days reduced the brood damages significantly. Measurements of the lithium concentrations in larvae and pupae during a chronic exposure with 10, 17.5 and 25 mM LiCl revealed respective lithium levels in 5th instar larvae of 7, 13 and 15 mg/kg. No lithium was detectable in 2-day old larvae indicating that pure worker jelly from the hypopharyngeal gland is not contaminated with LiCl. Based on these results, applications of LiCl in colonies with brood should be avoided.

Larvicidal activity of Artemisia absinthium extracts with special reference to inhibition of detoxifying enzymes in larvae of Aedes aegypti L

Insect-borne diseases continue to be a major source of sickness and death worldwide. Resistance to chemical pesticides and their risks have been regarded as a setback in mosquito vector control. Due to the presence of various phytocompounds in plant species, botanicals can manage and prevent vector (insect) transmitted illnesses by killing insect eggs and larvae. The purpose of this study was to determine the toxicity ofArtemisia absinthiumextracts (methanolic and ethanolic) against Aedes aegypti larvae (vector of arboviruses). Larval mortality was recorded after 24 h of exposure. Our findings showed that the ethanolic leaf extract ofArtemisia absinthiumhas higher larvicidal activity (92 ± 1.99 percent at 1000 ppm) than the methanolic extract (68 ± 1.99 percent at 1000 ppm). Furthermore, the ethanolic extract inhibited the activity of detoxifying enzymes acetylcholinesterase, α and β-carboxylesterases activity significantly. Besides, the protein level was also lowered upon exposure of ethanolic extract for 24 h against 4th instar larvae at an LC50 of 694.3 ppm. These findings clearly show that the Artemisia absinthium active extract (ethanolic) can be utilized as a pest and vector control option, helping to prevent the spread of infectious illnesses.

Antifeedant Mechanism of Dodonaea viscosa Saponin A Isolated from the Seeds of Dodonaea viscosa.

Dodonaea viscosa is a medicinal plant which has been used to treat various diseases in humans. However, the anti-insect activity of extracts from D. viscosa has not been evaluated. Here, we found that the total saponins from D. viscosa (TSDV) had strong antifeedant and growth inhibition activities against 4th-instar larvae of Spodoptera litura. The median antifeeding concentration (AFC50) value of TSDV on larvae was 1621.81 μg/mL. TSDV affected the detoxification enzyme system of the larvae and also exerted antifeedant activity possibly through targeting the γ-aminobutyric acid (GABA) system. The AFC50 concentration, the carboxylesterase activity, glutathione S-transferases activity, and cytochrome P450 content increased to 258%, 205%, and 215%, respectively, and likewise the glutamate decarboxylase activity and GABA content to 195% and 230%, respectively, in larvae which fed on TSDV. However, D. viscosa saponin A (DVSA) showed better antifeedant activity and growth inhibition activity in larvae, compared to TSDV. DVSA also exerted their antifeedant activity possibly through targeting the GABA system and subsequently affected the detoxification enzyme system. Further, DVSA directly affected the medial sensillum and the lateral sensillum of the 4th-instar larvae. Stimulation of Spodoptera litura. with DVSA elicited clear, consistent, and robust excitatory responses in a single taste cell.