Antifungal and Anti-Virulent Activity of Origanum majorana L. Essential Oil on Candida albicans and In Vivo Toxicity in the Galleria mellonella Larval Model.

The aim of this study was to investigate and compare in detail both the antifungal activity in vitro (with planktonic and biofilm-forming cells) and the essential oil composition (EOs) of naturally growing (OMN) and cultivated (OMC) samples of Origanum majorana L. (marjoram). The essential oil composition was analyzed using GC-MS. The major constituent of both EOs was carvacrol: 75.3% and 84%, respectively. Both essential oils showed high antifungal activity against clinically relevant Candida spp. with IC50 and IC90 less than or equal to 0.5 µg mL-1 and inhibition of biofilm with a concentration of 3.5 µg mL-1 or less. Cultivated marjoram oil showed higher anti-biofilm activity against C. albicans. In addition, OMC showed greater inhibition of germ-tube formation (inhibition by 83% in Spider media), the major virulence factor of C. albicans at a concentration of 0.125 µg mL-1. Both EOs modulated cell surface hydrophobicity (CSH), but OMN proved to be more active with a CSH% up to 58.41%. The efficacy of O. majorana EOs was also investigated using Galleria mellonella larvae as a model. It was observed that while the larvae of the control group infected with C. albicans (6.0 × 108 cells) and not receiving treatment died in the controls carried out after 24 h, all larvae in the infected treatment group survived at the end of the 96th hour. When the treatment group and the infected group were evaluated in terms of vital activities, it was found that the difference was statistically significant (p < 0.001). The infection of larvae with C. albicans and the effects of O. majorana EOs on the hemocytes of the model organism and the blastospores of C. albicans were evaluated by light microscopy on slides stained with Giemsa. Cytological examination in the treatment group revealed that C. albicans blastospores were phagocytosed and morphological changes occurred in hemocytes. Our results indicated that the essential oil of both samples showed strong antifungal activities against planktonic and biofilm-forming C. albicans cells and also had an influence on putative virulence factors (germ-tube formation and its length and on CSH).

Serine protease SP7 cleaves prophenoloxidase and is regulated by two serpins in Ostrinia furnacalis melanization.

Melanization is an innate immune response in insects to defend against the invading pathogens and parasites. During melanization, prophenoloxidase (PPO) requires proteolytic activation by its upstream prophenoloxidase-activating protease (PAP). We here cloned a full-length cDNA for a serine protease, named as SP7, from Ostrinia furnacalis. The open reading frame of SP7 encodes 421-amino acid residue protein with a 19-residue signal peptide. qRT-PCR analysis showed that SP7 mRNA levels were significantly upregulated upon exposure to microbial infection. Recombinant SP7 zymogen was activated by serine protease SP2. The active SP7 could cleave O. furnacalis PPOs including PPO2, PPO1b and PPO3. Additionally, active SP7 could form covalent complexes with serine protease inhibitor serpin-3 and serpin-4. The activity of SP7 in cleaving a colorimetric substrate IEARpNA or O. furnacalis PPOs was efficiently blocked by either serpin-3 or serpin-4. Our work thus revealed that SP7 and SP2 partially constituted a PPO activation cascade in which SP7 was activated by SP2 and then likely worked as a PAP. SP7 was effectively regulated by serpin-3 and serpin-4. The results would allow further advances in the understanding of melanization mechanisms in O. furnacalis.

Ethological aspects of Helicoverpa armigera in the reproductive phase of cotton

Pest behavior studies are important to inform the periods when the pest is more exposed to pesticide. This study aimed to evaluate the movement and feeding of Helicoverpa armigera larvae in the first three instars during the reproductive phase of cotton. First, larval behavior was evaluated with a completely randomized design in a 3 × 5 factorial design with eight replications. The treatments were the instars versus behaviors (crawling, resting, waving, eating and dropping-off). In addition, another 3 × 6 factorial design with eight replications was performed to evaluate the plant region most preferred by the caterpillars. The treatments were the instars versus six parts of the plant (abaxial surface, adaxial surface, on floral bud, internal surface of bract, petiole, and terminal growth). Then, the period of the day when caterpillars are more active was also evaluated by a 3 × 2 factorial essay. The treatments were the instars versus periods of the day. Eating and resting are the most frequent behaviors for all instars. The plant regions preferred by the caterpillars for crawling are on the floral bud and the adaxial face of the leaf. There is no preferred period of the day for eating, resting, waving or dropping-off. Caterpillars (until the third instar) prefer to walk in the morning and other behaviors can occur at any period. Therefore, sprays should preferably occur in the morning, and the insecticide drops/spray should reach the flower buds and the adaxial surface of the leaves to contaminate the larvae.

The microsporidium Nosema pyrausta as a potent microbial control agent of the beet webworm Loxostege sticticalis.

The microsporidium Nosema pyrausta is an important mortality factor of the European corn borer, Ostrinia nubilalis. The present study was aimed at N. pyrausta virulence testing to the beet webworm (BW), Loxostege sticticalis. This agricultural pest, L. sticticalis, was highly vulnerable to N. pyrausta. The parasite's spores were located in salivary glands, adipose tissue, and Malpighian tubules of the infected specimens. Infection was transmitted transovarially through at least 3 laboratory generations, in which BW fitness indices were lower than in the control, and moth emergence and fertility decreased prominently. Transovarial infection was most detrimental to female egg-laying ability, resulting in zero fertility in F3. When propagated in BW, the microsporidium tended to increase its virulence to L. sticticalis, as compared to the Ostrinia isolates. The parasite's ability to infect this host at low dosages and transmit vertically should guarantee its effective establishment and spread within BW populations. In conclusion, N. pyrausta is a promising agent against BW, which is a notorious polyphagous pest in Eurasia.

Early Warning of Resistance to Bt Toxin Vip3Aa in Helicoverpa zea.

Evolution of resistance by pests can reduce the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Because of the widespread resistance of Helicoverpa zea to crystalline (Cry) Bt toxins in the United States, the vegetative insecticidal protein Vip3Aa is the only Bt toxin produced by Bt corn and cotton that remains effective against some populations of this polyphagous lepidopteran pest. Here we evaluated H. zea resistance to Vip3Aa using diet bioassays to test 42,218 larvae from three lab strains and 71 strains derived from the field during 2016 to 2020 in Arkansas, Louisiana, Mississippi, Tennessee, and Texas. Relative to the least susceptible of the three lab strains tested (BZ), susceptibility to Vip3Aa of the field-derived strains decreased significantly from 2016 to 2020. Relative to another lab strain (TM), 7 of 16 strains derived from the field in 2019 were significantly resistant to Vip3Aa, with up to 13-fold resistance. Susceptibility to Vip3Aa was significantly lower for strains derived from Vip3Aa plants than non-Vip3Aa plants, providing direct evidence of resistance evolving in response to selection by Vip3Aa plants in the field. Together with previously reported data, the results here convey an early warning of field-evolved resistance to Vip3Aa in H. zea that supports calls for urgent action to preserve the efficacy of this toxin.

Artificial Diets With Different Protein Levels for Rearing Spodoptera frugiperda (Lepidoptera: Noctuidae).

Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) is a pest of great economic importance which can feed on more than 300 plant species. As it is polyphagous, its host plants may have variable physical and chemical constitutions. This may influence larval development, as protein and carbohydrate levels are important factors for adequate biological development. The aim of this study was to evaluate insect developmental parameters as well as to compare the food consumption of S. frugiperda larvae reared using diets with different protein levels under laboratory conditions. Three artificial diet formulations were used: one typically used for routine laboratory rearing, based on bean, wheat germ and brewer's yeast (D1); one containing half the original amount of protein (D2), and the other with twice the original amount of protein (D3). The relative consumption rate (RCR), relative growth rate (RGR), and efficiency of conversion of ingested food (ECI) for S. frugiperda fourth instar larvae varied among diets. The protein present in the diet influenced the duration of larval and pupal periods and pupal weight, but did not affect larval survival, fecundity and longevity of adults. The different protein levels in the diets did not negatively influence population growth, so these three diet variations can be used for mass rearing in the laboratory. However, the influence of these diets on successive generations of the insect remains untested.

Identification and expression characteristics of putative chemosensory proteins in the peach fruit borer Carposina sasakii Matsumura (Lepidoptera: Carposinidae).

Chemosensory proteins (CSPs) are important for insect chemoreception, which bind, solubilize and transport hydrophobic chemical molecules from external environment to dendrite membrane of chemosensory neurons. Moreover, CSPs are also involved in non-sensory physiological activities. The peach fruit borers Carposina sasakii Matsumura (Lepidoptera: Carposinidae) seriously damage fruit trees and their chemoreception mainly occurs in the adult stage. We identified 10 putative CSPs (CsasCSP1 ~ CsasCSP10) from head transcriptomes of C. sasakii adult males and females, all of which are classic CSPs that have 4 conserved cysteines with a spacing pattern C1-X6-C2-X17-18-C3-X2-C4. Their phylogenetic characteristics were also described. An analysis using fluorescence quantitative PCR showed CsasCSP2 has the highest level of expression in the heads, so it is more likely to be involved in C. sasakii chemoreception than the other C. sasakii CSPs. CsasCSP1, CsasCSP3, CsasCSP4, CsasCSP6, CsasCSP7 and CsasCSP8 are expressed dominantly in the wings; CsasCSP5 and CsasCSP10 have the highest expression level in the thoraxes; CsasCSP9 is dominantly and equally expressed in the thoraxes and abdomens. This study contributes to understanding physiological functions of C. sasakii CSPs and chemosensory mechanism at C. sasakii molecular level.

Environmental variables influence the developmental stages of the citrus leafminer, infestation level and mined leaves physiological response of Kinnow mandarin.

Climate change has not only exacerbated abiotic stress, but has also rendered external conditions more feasible for pests to spread and infest citrus fruit. Citrus leafminer (Phyllocnistis citrella) is a potential pest that directly feeds the newly sprouted leaves and twigs of all three spring, summer and autumn flushes. Increasing temperatures in spring and autumn, leafminer accrued more heat units or developmental degree days to accelerate the biological stages of its life-cycle, thereby increasing the pressure of infestation. Present work was conducted at three different environmental conditions in Sargodha, Toba Tek Singh (TTS) and Vehari districts of the Punjab province, Pakistan; all three experimental sites were located in different agro-ecological zones. More infestation was recorded in all three flushes at TTS and Vehari than in Sargodha. Overall, more damage was observed due to higher temperatures in TTS and Vehari than in Sargodha. After May-June heat stress, spontaneous vegetative growth continued from July to November, produced newly spouted tender leaves for feeding the leafminer larvae, and was seen more in TTS and Vehari. Leafminer larva prefers to enter young and tender leaves with a maximum entrance in leaves up to 1 cm2 in size while observing no entrance above 3 cm2 of leaf size. Physiological response of leaves primarily attributed to chlorophyll and carotenoid contents, both of which were recorded lower in the mined leaves, thereby reducing leaf photosynthetic activity. Similarly, lower levels of polyphenols and antioxidant activity were also recorded in the mined leaves. The on-tree age of mined leaves of three vegetative flushes of Kinnow plant was also less counted than non-mined leaves. Climate change has affected vegetative phenology and become feasible for pests due to extemporaneous leaf growth, particularly leafminer, and eventually causes economic loss by supplying low carbohydrates either to hanging fruits or next-season crops.

Investigation of antibiofilm activity, antibacterial activity, and mechanistic studies of an amphiphilic peptide against Acinetobacter baumannii.

Biofilm-producing pathogens, such as Acinetobacter baumannii, have aroused escalating attention. Because these bacteria could secrete mixture with close-knit architecture and complicated components to resist traditional antibiotics. Here, we reported an amphiphilic peptide denoted as zp3 (GIIAGIIIKIKK-NH2), which showed favorable bioactivity against Acinetobacter baumannii ATCC 19606 (minimal inhibitory concentration, MIC = 4 µM) and low cytotoxicity to mammalian cells Vero (half maximal inhibitory concentration, IC50 > 100 µM). Importantly, zp3 could inhibit the formation of biofilm at micromole level and eliminate around 50% preformed biofilm at 32 µM after 6 h treatment. This peptide was able to bind with biofilm while maintaining a helical structure in a mimic biofilm-rich environment. In vivo test demonstrated that zp3 rescued 33.3% of larvae after 48 h infection and reduced 1 log live bacteria inside the animal body after 6 h treatment. The bactericidal mode for zp3 was attributed to the combination of influencing ions balance at low concentration and inducing permeability alteration and pore formation on the Acinetobacter baumannii membrane at high concentration. Application on medical textiles also proved that zp3 could perform a good antibacterial activity in practice.

RNAi-mediated silencing of vitellogenin gene curtails oogenesis in the almond moth Cadra cautella.

Vitellogenins, major yolk protein precursors, play an essential role in the reproduction and spread of all oviparous species, including insects. To investigate reproductive strategies of the warehouse moth Cadra cautella at the molecular level, a partial transcript of the C. cautella vitellogenin (CcVg) gene was extended through the rapid amplification of cDNA ends PCR and sequenced. The complete CcVg mRNA transcript was 5,334 bp long, which encoded a protein of 1,778 amino acids, including the first 14 amino acids of the signal peptide. The deduced CcVg protein contained a putative cleavage site (RTRR) at the amino-terminal side, similar to several other insect species. DGQR and GI/LCG motifs were present at the CcVg gene C-terminus, followed by nine cysteine residues. CcVg harbored 131 putative phosphorylation sites, numbering 84, 19, and 28 sites for serine, threonine, and tyrosine, respectively. The transcript showed a great resemblance with other lepidopteran Vgs. CcVg protein analysis revealed three conserved regions: 1) vitellogenin-N domain, 2) DUF 1943 (domain of unknown function), and 3) a von Willebrand factor type D domain. Additionally, sex, stage-specific, and developmental expression profiles of the CcVg gene were determined through RT-PCR. The Vg was first expressed in 22-day-old female larvae, and its expression increased with growth. The phylogenetic analysis based on different insect Vgs revealed that the CcVg exhibited close ancestry with lepidopterans. The CcVg-based RNAi experiments were performed, and the effects were critically evaluated. The qRT-PCR results showed that CcVg-based dsRNA suppressed the Vg gene expression up to 90% at 48 h post-injection. Moreover, CcVg-based RNAi effects resulted in low fecundity and egg hatchability in the CcVg-based dsRNA-treated females. The females laid eggs, but because of insufficient yolk protein availability the eggs could not succeed to hatch. The significant difference in the fecundity and hatchability unveils the importance of CcVg gene silencing and confirmed that the Vg gene plays a key role in C. cautella reproduction and it has the potential to be used as a target for RNAi-mediated control of this warehouse pest.

The steroid hormone 20-hydroxyecdysone counteracts insulin signaling via insulin receptor dephosphorylation.

The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph.

Plant-herbivorous insect networks: who is eating what revealed by long barcodes using high-throughput sequencing and Trinity assembly.

Interactions between plants and insects are among the most important life functions for all organism at a particular natural community. Usually a large number of samples are required to identify insect diets in food web studies. Previously, Sanger sequencing and next generation sequencing (NGS) with short DNA barcodes were used, resulting in low species-level identification; meanwhile the costs of Sanger sequencing are expensive for metabarcoding together with more samples. Here, we present a fast and effective sequencing strategy to identify larvae of Lepidoptera and their diets at the same time without increasing the cost on Illumina platform in a single HiSeq run, with long-multiplex-metabarcoding (COI for insects, rbcL, matK, ITS and trnL for plants) obtained by Trinity assembly (SHMMT). Meanwhile, Sanger sequencing (for single individuals) and NGS (for polyphagous) were used to verify the reliability of the SHMMT approach. Furthermore, we show that SHMMT approach is fast and reliable, with most high-quality sequences of five DNA barcodes of 63 larvae individuals (54 species) recovered (full length of 100% of the COI gene and 98.3% of plant DNA barcodes) using Trinity assembly (up-sized to 1015 bp). For larvae diets identification, 95% are reliable; the other 5% failed because their guts were empty. The diets identified by SHMMT approach are 100% consistent with the host plants that the larvae were feeding on during our collection. Our study demonstrates that SHMMT approach is reliable and cost-effective for insect-plants network studies. This will facilitate insect-host plant studies that generally contain a huge number of samples.

A gene silencing of V-ATPase subunit A interferes with survival and development of the tomato leafminer, Tuta absoluta.

RNA interference (RNAi) technology is not only considered as a tool to analyze gene function, but it is also potentially considered as a strategy to develop novel biopesticide. In the current study, a double-stranded RNA specific to v-ATPase subunit A of the tomato leafminer, Tuta absoluta (Meyrick; Lepidoptera: Gelechiidae), was orally administered. A gradual decrease in the expression of the gene was observed from Day 1 to 3 and resulted in significant larval mortality. These results suggest that v-ATPases A can be considered as a promising target gene by RNAi technology to be used in the management of the tomato leafminer.

Characterization and biocontrol potential of a naturally occurring isolate of Metarhizium pingshaense infecting Conogethes punctiferalis.

An entomopathogenic fungus was isolated from an infected larva of Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera), a highly polyphagous pest recorded from more than 120 plants and widely distributed in Asia and Oceanic countries. The fungus was identified as Metarhizium pingshaense Q.T. Chen & H.L. Guo (Ascomycota: Hypocreales) based on morphological characteristics and molecular studies. Scanning electron microscopic studies were conducted to study the infection of C. punctiferalis by M. pingshaense. Bioassay studies with purified conidial suspension proved that the isolate was highly virulent to C. punctiferalis, causing more than 86 % mortality to fifth instar larvae at 1 × 108 spores/mL, under laboratory conditions. The median lethal concentration (LC50) of the fungus against late instar larvae was 9.1 × 105 conidia/mL and the median survival time (MST) of late instar larvae tested at the doses of 1 × 108 and 1 × 107 conidia/mL were 4.7 and 6.4 days, respectively. The optimal temperature for fungal growth and sporulation was found to be 25 ± 1 °C. This is the first report of M. pingshaense naturally infecting C. punctiferalis. Isolation of a highly virulent strain of this fungus holds promise towards development of a potential mycoinsecticide against this pest.

RNA silencing of hormonal biosynthetic genes impairs larval growth and development in cotton bollworm, Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera, is a highly polyphagous pest, causing enormous losses to various economically important crops. The identification and in vitro functional validation of target genes of a pest is a prerequisite to combat pest via host-mediated RNA interference (RNAi). In the present study, six hormonal biosynthesis genes of H. armigera were chosen and evaluated by feeding insect larvae with dsRNAs corresponding to each target gene, viz., juvenile hormone acid methyltransferase (HaJHAMT), prothoracicotropic hormone (HaPTTH), pheromone biosynthesis-activating peptide (HaPBAP), molt regulating transcription factor (HaHR3), activated protein 4 (HaAP-4) and eclosion hormone precursor (HaEHP). The loss of function phenotypes for these hormonal genes were observed by releasing second instar larvae on to artificial diet containing target gene-specific dsRNAs. Ingestion of dsRNAs resulted in mortality ranging from 60% to 90%, reduced larval weight, phenotypic deformities and delayed pupation. The quantitative real-time PCR (qRT-PCR) analysis showed that the target gene transcript levels were decreased drastically (31% to 77%) as compared to control or unrelated control (GFP-dsRNA), and correlated well with the mortality and developmental defects of larvae. Also, a comparison of the silencing efficacy of un-diced long HaPTTH -dsRNAwith RNase III diced HaPTTH-dsRNA (siRNAs) revealed that long dsRNAs were more efficient in silencing the target gene. These results indicated that the hormonal biosynthesis genes have varied sensitivity towards RNAi and could be the vital targets for insect resistance in crop plants like cotton which are infested by H. armigera.

Synthesis, Insecticidal Activities, and Structure-Activity Relationship of Phenylpyrazole Derivatives Containing a Fluoro-Substituted Benzene Moiety.

Fluorinated organic compounds represent a growing and important family of commercial chemicals. Introduction of fluorine into active ingredients has become an effective way to develop modern crop protection products. Given the particular properties of fluorine and high efficiency and selectivity of diamide insecticides, we designed and synthesized 27 anthranilic diamides analogues containing fluoro-sustituted phenylpyrazole. A preliminary bioassay indicated that most target compounds exhibited good biological activity against Mythimna separata and Plutella xylostella. Compound IIIf containing a 2,4,6-trifluoro-substituted benzene ring showed 43% insecticidal activity against M. separata at 0.1 mg L-1, while the control chlorantraniliprole was 36%. The activity of IIIe against P. xylostella at 10-5 mg L-1 was 94%, compared with that of the control being 70%. Thus, introduction of fluorine into diamide insecticides was useful for increasing activity. Insect electrophysiology studies showed that the calcium concentration in the nerve cells of third M. separata larvae was elevated by IIIf, which further confirmed that ryanodine receptor (RyR) was its potential target.

Determination of Cotton as a Larval Feeding Source for Lepidopteran Moths Using a Derivative from Cotton Metabolites as a Marker by LC-MS/MS Method.

Determination of the feeding history of polyphagous insect pests, such as noctuid moths (Lepidoptera: Noctuidae), is a critical element in developing population and resistance management strategies for such pests. To identify reliable markers for larval host plant determination and to develop simple extraction and detection methods, a metabolomics approach was implemented after acid hydrolysis of adult moth samples. We identified a derivative from cotton metabolites as a marker in adult moths that were fed cotton tissues as a larval diet, and we propose that the marker is tricycloheliocide H4 based on NMR and mass fragmentation analysis. Using this derivative from cotton metabolites as a marker, a targeted LC-MS/MS method reliably identified cotton as a larval diet in extracts of three noctuid moth species: Helicoverpa zea (cotton bollworm), Chloridea (Heliothis) virescens (tobacco budworm) and Chrysodeixis includens (soybean looper). We are using similar approaches to identify markers for other host plants including soybean.

Characterization and expression analysis of genes encoding three small heat shock proteins in the oriental armyworm, Mythimna separata (Walker).

Small heat shock proteins (sHsps) function in the response of insects to abiotic stress; however, their role in response to biotic stress has been under-investigated. Mythimna separata, the oriental armyworm, is polyphenetic and exhibits gregarious and solitary phases in response to high and low population density, respectively. In this study, three genes were identified encoding sHsps, namely MsHsp19.7, MsHsp19.8 and MsHsp21.4, and expression levels in solitary and gregarious M. separata were compared. The deduced protein sequences of the three MsHsps had molecular weights of 19.7, 19.8 and 21.4 kDa, respectively, and contained a conserved α-crystalline domain. Real-time PCR analyses revealed that the three sHsps were transcribed in all developmental stages and were dramatically up-regulated at the 6th larval stage in gregarious individuals. Expression of the three MsHsps was variable in different tissues of 6th instar larvae, but exhibited consistent up- and down-regulation in the hindgut and Malpighian tubules of gregarious individuals, respectively. In addition, MsHsp19.7 and MsHsp19.8 were significantly induced when solitary forms were subjected to crowding for 36 h, but all three MsHsps were down-regulated when gregarious forms were isolated. Our findings suggest that population density functions as a stress factor and impacts MsHsps expression in M. separata.

Acting target of toosendanin locates in the midgut epithelium cells of Mythimna separate Walker larvae (lepidoptera: Noctuidae).

Toosendanin (TSN), which is extracted from the root bark of Melia toosendan Siebold and Zuccarini, has multiple modes of action against insects. Especially, this compound has a potent stomach poisoning activity against several lepidoptera pests. In this paper, the signs of toxicity, digestive enzymes activity, the histopathological changes and immuno-electron microscopic localization of TSN in the midgut epithelium of Mythimna separate Walker larvae were investigated for better understanding its action mechanism against insects. The bioassay results indicated that TSN has strong stomach poisoning against the fifth-instar larvae of M. separata (LC50 = 252.23 µg/mL). The typical poisoned symptom were regurgitation and paralysis. Activities of digestive enzymes had no obvious changes after treatment with LC80 dose of TSN. The midgut epithelial cells of insect were damaged by TSN, showing the degeneration of microvilli, hyperplasia of smooth endoplasmic reticulum and condensation of chromatin. Immunohistochemical analysis revealed that the gold particles existed on the microvilli of columnar cells and goblet cells, and gradually accumulated with the exacerbation of poisoning symptoms, showing that TSN targets on the microvilli of the midgutcells. Therefore, TSN acts on digestive system and locates in the microvilli of midgutcells of M. separata.

The development of an improved artificial diet for the mass rearing of Eldana saccharina Walker (Lepidoptera: Pyralidae).

BACKGROUND: This study determined the nutrient requirements of Eldana saccharina Walker (Lepidoptera: Pyralidae), a serious sugarcane pest in South Africa, to develop a more efficient artificial diet for mass-rearing purposes for sterile moth production. Diets tested consisted of a minimum specification (MS) diet representing a diet formulated according to the minimum specification of a summary of published diets, which yielded satisfactory results; an ideal amino acid profile (IAAP) diet, where amino acid composition was based on the profile of amino acids in the 2nd (IAAP2) and 5th /6th (IAAP5/6) instar larvae; and lastly two diets based on the nutrient composition of the natural diet of the insects, papyrus (PAP) and sugarcane (SC). Six treatments with 50 replications were randomly allotted to 300 25 mL plastic screw-top vials. The diet (15 mL) was dispensed into each vial and inoculated with two freshly hatched larvae. Larvae, pupae, and moths were harvested at 28 days after inoculation. Overall survivability, pupal weight, sex ratio, and rate of development was determined and compared with the diet currently in use at the South African Sugarcane Research Institute (CON). Physical characteristics of the diets such as the pH and the water-holding capacity of the diets were also determined. RESULTS: The natural diets (PAP and SC) were not viable as they did not yield any results. Survivability was significantly higher (78%) for the MS diet whilst IAAP2 and IAAP5/6 yielded the second highest survivability (74%) compared to CON (68%). There were no differences in male pupal weights between all treatment diets, as was the case for female pupae. Within dietary treatments, female pupae were heavier than male pupae for all treatment diets. CON (1.0: 1.6) produced significantly less male than female pupae with MS (1.0: 1.2), IAAP2 (1.0: 1.0) and IAAP5/6 (1.0: 1.1) all producing equal amounts of male and female pupae. The MS diet (16%) yielded fourfold the number of moths after 28 days compared to CON (4%) and IAAP2 (4%) diets. IAAP5/6 yielded no moths after 28 days. The life stages thus developed fastest in the MS diet. The pH of all treatment diets remained stable for the entire duration of the trial. No biological contamination was observed through all diets. Differences in water-holding capacity were observed between most diets with PAP and SC losing the most moisture whilst the MS and IAAP2 diets retained the most moisture. CONCLUSION: The MS diet most closely represented the nutrient requirements of E. saccharina, leading to its faster development on this formulation, which could be readily applied for large-scale production of this lepidopteran pest as an aid in the mass rearing of sterile males as part of the integrated pest management plan. © 2020 Society of Chemical Industry.