Larvae Of Pieris Brassicae Research Articles

Investigation of Changes in Expression Levels of Detoxification Genes and Mortality in Pieris brassicae Larvae Exposed to Nepeta meyeri

Investigation of Changes in Expression Levels of Detoxification Genes and Mortality in Pieris brassicae Larvae Exposed to Nepeta meyeri

Amending soil with insect exuviae improves herbivore tolerance, pollinator attraction and seed yield of Brassica nigra plants

To promote circularity in agriculture, the residual –streams from the production of insects as feed and food, such as insect exuviae (moulted skins), can be a sustainable novel organic soil amendment for crop production. Organic soil amendments can influence soil composition by providing nutrients and stimulating the growth of beneficial microorganisms. Soil composition significantly impacts plant growth and resistance against herbivores. However, little is known about the effect of soil composition on flower visitors and seed production. Here, black soldier fly (BSF; Hermetia illucens) exuviae was added to soil to investigate the effects on plant growth, plant resistance against two insect herbivores, and the consequences for attraction of pollinators and seed yield. Brassica nigra seeds were sown in field soil or in field soil mixed with powdered BSF exuviae. Three-week-old plants and the soil of each pot were planted in a common garden. The effects on vegetative and flower traits, interactions with flower visitors and seed yield were quantified. In addition, the performance of the leaf-chewing larvae of Pieris brassicae and the piercing-sucking aphid Brevicoryne brassicae were assessed. Brassica nigra plants grown on soil amended with BSF exuviae showed increased growth, had more flowers and flower visitors, and higher seed production. Herbivory neither affected number of flowers nor seed production, suggesting tolerance and compensatory growth responses to herbivory compared to plants growing in soil without the addition of BSF exuviae. After five weeks fewer aphids were found on plants growing in amended soil. Our findings show that BSF exuviae added to soil can positively affect tolerance to herbivory and seed production of B. nigra, an insect-pollinated plant species. Moreover, adding insect exuviae as soil amendment in the field benefits plant-pollinator mutualism and seed yield even during herbivore attack in the field. Use of by-products from insect production as soil amendment can contribute to sustainable agricultural practices along with the conservation of ecosystem services.

Virulence potential of two entomopathogenic nematodes, their associated bacteria, and its metabolites to larvae of Pieris brassicae L. (Lepidoptera, Pieridae) in cabbage under greenhouse and field bioassays

Virulence potential of two entomopathogenic nematodes, their associated bacteria, and its metabolites to larvae of Pieris brassicae L. (Lepidoptera, Pieridae) in cabbage under greenhouse and field bioassays

Insect egg deposition renders plant defence against hatching larvae more effective in a salicylic acid-dependent manner.

Plants can improve their antiherbivore defence by taking insect egg deposition as cue of impending feeding damage. Previous studies showed that Pieris brassicae larvae feeding upon egg-deposited Brassicaceae perform worse and gain less weight than larvae on egg-free plants. We investigated how P.brassicae oviposition on Arabidopsis thaliana affects the plant's molecular and chemical responses to larvae. A transcriptome comparison of feeding-damaged leaves without and with prior oviposition revealed about 200 differently expressed genes, including enhanced expression of PR5, which is involved in salicylic acid (SA)-signalling. SA levels were induced by larval feeding to a slightly greater extent in egg-deposited than egg-free plants. The adverse effect of egg-deposited wild-type (WT) plants on larval weight was absent in an egg-deposited PR5-deficient mutant or other mutants impaired in SA-mediated signalling, that is, sid2/ics1, ald1, and pad4. In contrast, the adverse effect of egg-deposited WT plants on larvae was retained in egg-deposited npr1 and wrky70 mutants impaired further downstream in SA-signalling. Oviposition induced accumulation of flavonols in WT plants with and without feeding damage, but not in the PR5-deficient mutant. We demonstrated that egg-mediated improvement of A.thaliana's antiherbivore defence involves SA-signalling in an NPR1-independent manner and is associated with accumulation of flavonols.

Releasing Digestive Enzymes by Brain Peptides in the Larvae of Pieris brassicae L. (Lepidoptera: Pieridae)

Neuropeptides synthesized by insect neurosecretory cells are engaged in several physiological process like immunity, feeding and reproduction so they deserve to be undertaken in subsequent experiments for discovery and manipulation regarding applied objectives. Due to importance of insect ability to utilize plant tissues, the present study was conducted to extract brain peptides in the fifth instar larvae of Pieris brassicae via High performance liquid chromatography and to identify their role in induction of digestive enzyme syntheses. Five peaks were determined and named as fractions 1, 2, 3, 4 and 5. The midguts of starved fifth larval instars were treated by each fraction of brain extract in vitro. The highest activities of α-amylase and lipase were found in the midgut treated by fractions 4 and 1, respectively while the fraction number 5 imposed the highest activities of glucosidases. Fraction 2 led to the highest activities of trypsin, chymotrypsin and elastase as the given serine proteinases as well as the two exopeptidases namely amino- and carboxypeptidases. Moreover, midgut treatments by different concentrations of TLCK (N-p-Tosyl-l-lysine methyl ester hydrochloride), TPCK (Tosyl phenylalanyl chloromethyl ketone), AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) and Acarbose significantly decreased the activities of α-amylase, trypsin, chymotrypsin and elastase by the calculated IC50 values of 0.287, 0.439, 0.288 and 0.361 mM, respectively. The findings demonstrated that different neuropeptides would be engaged in enzymatic release of P. brassicae hence, manipulation of peptide receptors can change physiological process of insects and opens a selective strategy to pest control.

Immune defense of Pieris brassicae larvae in challenged with Heterorhabditis bacteriophora, its symbiotic bacteria and metabolites

In this study, virulence of the culture broth of Photorhabdus temperata subsp. temperata and its aqueous and organic extracts were investigated for 3rd and 4th instars of Pieris brassicae (Lepidoptera: Pieridae) larvae. Virulent efficacy was found in all the treatments. However, the culture broth of bacteria showed greater mortality effect. Also, this study presents the response of P. brassicae immune system factors, phenoloxidase, lysozyme and hemocytes, to infection by Heterorhabditis bacteriophora, its symbiotic bacteria and their aqueous and organic extracts. Results showed that after injecting infective juveniles (IJs) with phenoloxidase, specific activity was suppressed after primary increase. However, the phenoloxidase specific activity suppression was observed earlier after injecting P. temperata subsp. temperata, aqueous and organic extracts. Also, results indicate more phenoloxidase activity of 4th instars larvae in comparison with 3rd instars larvae, which can show higher activity of phenoloxidase in the 4th larval stage. After injecting IJs into hemocel, the total hemocyte count was increased until five h post-injection and then decreased at seven hours post-injection. Differential Hemocyte Count (DHC) indicated granolucytes and plasmocytes as frequent hemocytes after treatment. Lysozyme consideration showed an increase after injecting living and heat-killed bacteria into hemocel.

Studies of the effects of imidacloprid and deltamethrin insecticides on the biology of Pieris brassicae (Linn.) on cabbage

The present investigation was conducted during Rabi season of 2011 to see the effect of imidacloprid and deltamethrin insecticides on the biology of Pieris brassicae (Linn.) on cabbage at experimental field of the Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh. The larvae of Pieris brassicae were collected from cabbage field in the month of January and February 2012. They were kept in jars measuring 25 x15 cm and provided fresh cabbage leaves as food for larvae. These jars were kept in B.O.D chamber at 24.5 oC and 70±10 per cent relative humidity. Fresh cabbage leaves were dipped in 0.05 per cent aqueous solution of, imidacloprid and deltamethrin and dried at room temperature by keping in the experimental jars. Fifteen third instar larvae in a batch of five were allowed to feed on them for 24 hours. Survivorship and expectancy of the life was greatest in the beginning of the age and decreased gradually with the advancement of the age in the all treatment including control. The expectancy was minimum (17.18) when P. brassicae was treated with imidacloprid while maximum in the untreated individuals (25.83). The mortality of P. brassicae was significantly high in the early instars as compared to late instars in both treated and untreated individuals. How to view point the article : Khan, Hadi Husain, Ansari, M. Shafiq, Hasan, Fazil, Ahmad, Salman and Danish, Mohd. (2016). Studies of the effects of imidacloprid and deltamethrin insecticides on the biology of Pieris brassicae (Linn.) on cabbage. Internat. J. Plant Protec., 9(1) : 313-321.

Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae).

The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones.

Expression of prtA from Photorhabdus luminescens in Bacillus thuringiensis enhances mortality in lepidopteran larvae by sub-cutaneous but not oral infection

The prtA gene from Photorhabdus luminescens encodes the virulence factor Protease A. When P. luminescens is injected into the hemocoel of insects by entomopathogenic nematodes, PrtA is a key component of pathogenicity thought to help degrade the immune system. The prtA gene was cloned and introduced on a plasmid into Bacillus thuringiensis. PrtA was shown to be actively expressed in vitro by cleavage of a specific Dabcyl–Edans heptapeptide substrate. There was no difference in the speed or level of mortality when spores and δ-endotoxins crystals of the transformed strain were fed to larvae of Pieris brassicae, as compared to the wild-type strain. When vegetative cells were injected into the hemocoel of larvae of Galleriamellonella, however, there was a significant increase in the rate and level of mortality over the wild type. The yield of B. thuringiensis per cadaver was a hundred-fold greater in the PrtA-secreting strain. The increased pathogenicity from intrahemocoelic infection may have been due to a greater ability to overcome the immune response of G. mellonella while other factors such as resident gut bacteria may have negated this advantage after oral dosage.

Density‐dependent investment in costly anti‐predator defences: an explanation for the weak survival benefit of group living

A central explanation for group living across animal taxa is the reduced rate of attack by predators. However, many field observations show a weak or non-existent effect of group size on per capita mortality rates. Herein we resolve this apparent paradox. We found that Pieris brassicae larvae defended themselves less readily when in groups than when alone, in that they were more reluctant to regurgitate in response to simulated attacks and produced less regurgitant. Furthermore, a simple model demonstrates that this reluctance was sufficient to cancel out the benefit from being in a group. This conditional strategy can be understood in terms of the costs and benefits of defences. For grouped individuals, defence is less often required because attack rates are lower and the costs of defence may be higher due to competition for resources. These phenomena are likely to be widespread in facultatively gregarious species that utilise anti-predator defences.

Invertebrate pathogenicity and toxin-producing potential of strains of Bacillus thuringiensis endemic to Antarctica

Several strains of Bacillus thuringiensis were previously isolated from soil in Antarctica and appeared to have physiological adaptations to this cold, nutrient-poor environment. In spite of this they could produce abnormally large, parasporal crystals under laboratory conditions. Here, they have been further characterised for toxin genes and invertebrate pathogenicity. All of the strains were positive in PCR assays for the cry1Aa and cry2 genes. This was confirmed by sequence analysis and the parasporal crystals of all strains contained polypeptides of about 130kDa. This potential for lepidopteran toxicity was borne out in bioassays of purified δ-endotoxins against larvae of Pieris brassicae: the LD50 values of B2408 (288μg) were comparable to that of the reference strain, HD-12 (201μg). There was no activity against the nematode Caenorhabditis elegans in spite of the fact that all strains appeared to possess the cry6 gene. PCR screening for genes encoding other nematode-toxic classes of toxins (Cry5, 4 and 21) was negative. B. thuringiensis has never previously been shown to be toxic to Collembola (springtails) but the purified δ-endotoxins of one of the Antarctic strains showed some activity against Folsomia candida and Seira domestica (224μg and 238μg, respectively). It seems unlikely that the level of toxicity demonstrated against springtails would support a pathogenic life-style in nature. All of the strains were positive for genes encoding Bacillus cereus-type enterotoxins. In the absence of higher insects and mammals the ecological value of retaining the toxic capability demonstrated here is uncertain.

Toxic effects of neem-based insecticides on Pieris brassicae (Linn.)

Four neem-based insecticides, Neemix ® (0.25% EC @ 20 mg azadirachtin/liter), Ecozin ® (3% EC @ 20 mg azadirachtin/liter), Agroneem ® (0.15% EC @ 4.8 mg azadirachtin/liter) and Neem oil (0.25% EC azadirachtin @ 20 mg azadirachtin/liter) and a non-commercial neem leaf powder, were evaluated for oviposition deterrence, antifeedant effect to larvae and toxicity to eggs and larvae of Pieris brassicae (Linn.) on cabbage leaves in the laboratory. The concentrations tested were with in the ranges of recommended field rates. Oviposition deterrence in no-choice, two-choice and six-choice assays, was observed for all the treatments. They exhibited significant ( P < 0.01) oviposition deterrence on P. brassicae when compared with a non-treated control. Cabbage leaves treated with the neem-based insecticides were used as an egg-laying substrate. Numbers of eggs oviposited by P. brassicae adults on treated cabbage leaves were significantly lower then those treated with water, but no significant differences were detected among the neem insecticides. They also deterred feeding by Pieris larvae and exhibited significant antifeedant effects. Larvae of P. brassicae on treated leaves stopped feeding and dropped from the leaf, resulting in no or minimal damage. Direct contact with neem-based insecticides decreased the survival of eggs. Survival of larvae fed for 9 days on leaves treated with neem-based insecticides was reduced to 51%, 49%, 48%, 24% and 18% in the Neem oil, Neemix, Agroneem, Ecozin and neem leaf powder treatments, respectively. Therefore, it can be concluded from the present investigations that neem-based insecticides had oviposition deterrence, antifeedant and toxic effect to P. brassicae.

Predation capacity and prey preference of Chrysoperla carnea on Pieris brassicae

The predation capacity and prey preference of larvae of Chrysoperlacarnea (Stephens) (Neuroptera: Chrysopidae) on eggs or larvae of Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) in the absence and presence of cabbage aphids as an alternative prey were evaluated in laboratory experiments at 25°C. Both instars preyed upon butterfly eggs and larvae as well as on cabbage aphids with the third instar being the most voracious. The lacewings had a strong preference for caterpillars to butterfly eggs. In the presence of the aphids the predation on P. brassicae eggs or larvae was either completely abandoned or reduced by about 70%, respectively, by second instar lacewings and either reduced by about 80% or maintained, respectively, by third instar lacewings. Both instars thus had a clear preference for aphids compared to eggs of P. brassicae. However, second instar lacewings preferred aphids to caterpillars whereas the opposite was the case for third instar lacewings. The results indicate that 3rd instar C. carnea has a potential as biocontrol agent against P. brassicae.

Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72h. The bacteria multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass.

The Ecology of Bacillus thuringiensis on the Phylloplane: Colonization from Soil, Plasmid Transfer, and Interaction with Larvae of Pieris brassicae

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.

Pyrrolizidine alkaloids from Anchusa strigosa and their antifeedant activity

The pyrrolizidine alkaloid (PA) content of flowers, leaves, and roots of Anchusa strigosa (Boraginaceae) was analysed by ESI–LC–MS. Six PAs, including two new natural compounds, were detected, characterized by NMR spectroscopy, and quantified in each plant organ. The results indicated that the highest total concentration of PAs was in the leaves (23.63 mg/g of dried part), followed by the flowers (19.77 mg/g), and finally by the roots (1.80 mg/g). All PAs isolated were subjected to Spodoptera exigua and Pieris brassicae larvae. Feeding activity by both herbivore species using a bioassay was inhibited up to circa 75% depending on PA and applied concentration.

Response of the wasp (Cotesia glomerata) to larvae of the large white butterfly (Pieris brassicae)

The large white butterfly (Pieris brassicae L) first invaded northernmost Japan from Siberia around 1994, and after a few years, began to expand its range. The wasp, Cotesia glomerata (L) parasitizes larvae of the small white butterfly (Pieris rapae crucivora Boisduval), a usual host in the same geographic area. Some Pieris brassicae larvae in Hokkaido have been parasitized by Cotesia glomerata, but the parasitism rate of Pieris brassicae larvae tends to be lower than that of Pieris rapae. To examine the process of parasitizing Pieris brassicae larvae, we observed how the parasitoid wasp responded to the host larvae on damaged leaves. Cotesia glomerata females tended to avoid Pieris brassicae larvae, and even when female wasps inserted their ovipositors into Pieris brassicae larvae, none laid eggs. The parasitoids obtained from Pieris rapae larvae failed to parasitize Pieris brassicae during the host‐acceptance step.

Lack of sequestration of host plant glucosinolates in Pieris rapae and P. grarricae

Sequestration of plant toxins in herbivores is often correlated with aposematic coloration and gregarious behaviour. Larvae of Pieris brassicae show these conspicuous morphological and behavioural characteristics and were thus suggested to sequester glucosinolates that are characteristic secondary metabolites of their host plants. P. rapaeare camouflaged and solitary, and are thus not expected to sequester. To test this hypothesis and to check the repeatabi-lity of a study that did report the presence of the glucosinolate sinigrin in P. brassicae, larvae were reared on three species of Brassicaceae (Sinapis alba, Brassica nigra and Barbarea stricta), and different leaf and insect samples were taken for glucosinolate analysis. The major host plant glucosinolates could only be found in traces or not at all in larval haemolymph, bled or starved larvae, faeces or pupae of both species or P. brassicae regurgitant. Haemolymph of both Pieris spp. was not rejected by the ant Myrmica rubra in dual-choice assays; the regurgitant of P. brassicae was rejected. This suggests the presence of compounds other than glucosinolates that might be sequestered in or produced by P. brassicae only. In faeces of both Pieris spp. a compound which yielded 4-hydroxybenzylcyanide (HBC) upon incubation with sulfatase was detected in high concentrations when larvae had been reared on S. alba. This compound may be derived from hydrolysis of sinalbin, the main glucosinolate of that plant. The unidentified HBC progenitor was apparently not sequestered in the two Pieris spp., and was not detected in faeces of larvae reared on B. nigra or B. stricta.

Behavioural observations of Pieris brassicae larvae indicate multiple mechanisms of action of analogous drimane antifeedants

We tested 11 analogous synthetic drimane antifeedant compounds for their feeding inhibiting effects on larvae of the large white butterfly Pieris brassicae L. (Lepidoptera: Pieridae) in no-choice tests on the host plant Brassica oleracea L. Furthermore, we observed larval feeding behaviour in no-choice tests to analyze temporal effects of five drimanes. The results show that the five analogous antifeedants differentially influence feeding behaviour and locomotion activity. Warburganal and polygodial are most likely sensory mediated antifeedants. Habituation to these compounds occurs soon after the onset of the tests (i.e., within 0.5–1.5 h). Compound 5 and confertifolin are probably not direct, sensory mediated antifeedants. After 0.5–1.5 h of exposure, these compounds inhibit not only feeding, but also locomotion behaviour, indicating postingestive, toxic effects. Isodrimenin inhibits feeding from the onset of the test and is probably a sensory mediated antifeedant. No habituation occurs to this compound, indicating that isodrimenin is either a very strong antifeedant or that it additionally has postingestive, toxic effects. Topical application of the drimanes on the larval cuticle revealed feeding inhibiting effects, but these could not be related to the occurrence of postingestive feeding inhibiting effects, indicating that this method is inappropriate to show possible postingestive effects of drimanes in P. brassicae. In conclusion, the behavioural observations performed in this research indicate that analogous drimanes inhibit feeding by P. brassicae larvae through multiple mechanisms of action. The results show that, when developing a structure activity relationship (SAR) for a series of antifeedants, it is important to distinguish the mode of action which underlies inhibition of feeding.

Synthesis and insect antifeedant activity of 2-substituted 2,3-dihydrofuran-3-ols and butenolides (Part II)

The introduction of a hydroxy group at C-13 of the clerodane skeleton is proposed as a strategy to potentially increase the antifeedant activity of simple analogs. This idea is applied to clerodane analogs with furan or butenolide type sidechains and therefore syntheses were developed for several types of 3-alkyl-substituted butenolides, 3-alkyl-substituted-3-hydroxybutenolides and 3-hydroxytetrahydrofuran derivatives. The antifeedant activity was tested on 5th instar larvae of Pieris brassicae.