Aposematic and cryptic Lepidoptera tested on the mouse

The toxicity of 53 species of Lepidoptera which are aposematic (a) at all stages of development; (b) as larvae but not as adults; (c) as adults but not as larvae, is compared with that of cryptic species, by means of a qualitative test consisting of intraperitoneal injection into the laboratory mouse. Points of interest which emerged from this study include (i) Pieris brassicae is toxic whether or not the larval diet contains sinigrin and mustard oils; (ii) a marked sexual dimorphism in toxicity is noted in several species; (iii) a yellow (recessive) variety of Callimorpha dominula (ab. lutea ) is less toxic than the typical form; (iv) P. brassicae pupae are acceptable to certain birds if the larvae are reared on artificial diet, but rejected (on account of taste or smell) if fed on cabbage; (v) only Arctia caja , out of 14 species tested, retains its toxicity when dried, and (vi) the gravid female Zygaena trifolii and her eggs kill the mouse in two to three minutes.     

Distribution, Frequency, and Diversity of Bacillus thuringiensis in an Animal Feed Mill

Bacillus thuringiensis was isolated from 36 of 50 residue samples obtained from an animal feed mill (a stored-product environment). Of 710 selected colonies having Bacillus cereus-B. thuringiensis morphology isolated from the samples, 477 were classified as B. thuringiensis because of production of parasporal delta-endotoxin crystals. There was a diverse population of B. thuringiensis, as revealed by differentiation of the isolates into 36 subgroups by using (i) their spectra of toxicity to the lepidopterans Heliothis virescens, Pieris brassicae, and Spodoptera littoralis and the dipteran Aedes aegypti and (ii) their parasporal crystal morphology. A total of 55% of the isolates were not toxic to any of these insects at the concentrations used in the bioassays; 40% of all isolates were toxic to one or more of the Lepidoptera; and 20, 1, and 1% of the isolates were toxic to only P. brassicae, H. virescens, and S. littoralis, respectively. The most frequent toxicity was toxicity to P. brassicae (36% of all isolates); 18% of the isolates were toxic to A. aegypti (5% exclusively), 10% were toxic to H. virescens, and 4% were toxic to S. littoralis. Toxicity to P. brassicae was more often linked with toxicity to H. virescens than with toxicity to S. littoralis. The frequency of toxicity was significantly greater in isolates that produced bipyramidal crystals than in isolates that produced irregular pointed, irregular spherical, rectangular, or spherical crystals.     

Chemoreception of amino acids in larvae of two species of Pieris

ABSTRACT. Specificity and sensitivity of gustatory neurones in response to twenty-two amino acids were studied in larvae of Pieris brassicae L. and Pieris rapae L. (Lepidoptera: Pieridae) using electrophysiological methods. Twelve amino acids stimulated a specific amino acid receptor cell in the lateral styloconic sensillum on the maxillary galea of both species, and a further two evoked single unit responses in the same sensillum of P.brassicae only. Histidine, phenylalanine and tryptophane were the weakest stimulants for P.brassicae , but were among the four best stimulants for P.rapae . In both species, eight amino acids were ineffective. Significant differences in stimulatory effectiveness were found between amino acids. Nutritionally essential amino acids were more effective in both species, as in five other lepidopterous species. Similarities with postulated sites for amino acid recognition in the dipteran Boettcherisca peregrina were found.
Concentration-response (C/R) relations were studied for five amino acids. Significant differences were found in saturated response levels. Parameters characterizing C/R relations were estimated using a logistic model. Comparing C/R parameters with phytochemical data on concentrations of free amino acids in a common host plant, Brassica oleracea L., shows that amino acids are effective stimuli at their natural concentrations. The amino acid chemoreceptor seems able to transmit information about concentration differences of amino acids in the plant tissue.     

Tronchuda cabbage flavonoids uptake by Pieris brassicae

The flavonoid pattern of larvae of cabbage white butterfly (Pieris brassicae L.; Lepidoptera: Pieridae) reared on the leaves of tronchuda cabbage was analysed by HPLC-DAD-MS/MS-ESI. Twenty flavonoids were identified or characterised, namely 16 kaempferol and four quercetin derivatives. Kaempferol 3-O-sophoroside, a minor component of tronchuda cabbage, was found to be the main component in P. brassicae (15.8%). Apart from this, only two other flavonoids present in significant amounts in tronchuda cabbage (kaempferol 3-O-sophoroside-7-O-glucoside and kaempferol 3-O-sophoroside-7-O-sophoroside) were found in the larvae. The larvae have high amounts of quercetin derivatives (18.5%), which were present only in trace amounts in tronchuda cabbage extracts, suggesting that P. brassicae is able to selectively sequester these flavonoids. The occurrence of a high content of flavonoids not detectable in tronchuda cabbage extracts indicates that P. brassicae larvae are able to metabolize dietary flavonoids.     

Carotenoids of the pupae of the Large White butterfly (Pieris brassicae) and the Small White butterfly (Pieris rapae)

The light cues received by the larvae of Pieris brassicae which determine diapause can also influence the carotenoid distribution (and hence the colour) in the epidermis and cuticle of the diapausing pupae. Irrespective of background or light cues received during the "sensitive period" of the pharate pupa, these diapausing pupae are coloured green. They then contain more than double the concentration of carotenoids in their epidermis than the non-diapausing pupae. This green colour can be somewhat modified by switching the full grown larvae to long day regimes immediately after feeding ceases.
The Large White and Small White butterfly each has a characteristic carotenoid storage pattern, which can be demonstrated by feeding the larvae on portions of the same cabbage leaves. The braconid parasite Apanteles glomeratus mirrors the carotenoids in its host.     

Relationships between parasitoid host range and host defence: a comparative study of egg encapsulation in two related parasitoid species

Abstract. Parasitoid host range may proceed from traits affecting host suitability, traits affecting parasitoid foraging behaviour, or both. We tested the hypothesis that encapsulation can be used as a reliable indicator of parasitoid host range in two closely related larval endoparasitoids of Lepidoptera. Cotesia glomerata (L.) (Hymenoptera: Braconidae) is gregarious and a generalist on several species of Pieridae, whereas C. rubecula (Marshall) is solitary and specific to Pieris rapae (L.). We determined the effects of host species ( Pieris brassicae (L.), P. napi (L.) and P. rapae ) (Lepidoptera: Pieridae) and host developmental stage (early first, second and third instar) on encapsulation of parasitoid eggs. Host species and parasitoid species, as well as the resulting interaction between these two factors had significant effects on encapsulation of Cotesia eggs. Encapsulation in Pieris hosts was much lower for C. glomerata (<34%, except for second and third instar of P. rapae ) than for C. rubecula (>32%), even when the latter was parasitizing P. rapae. Encapsulation increased with the age of the larvae, although the only significant difference was for C. glomerata. Overall, P. rapae showed a stronger encapsulation reaction than P. brassicae and P. napi. Encapsulation levels of C. glomerata corresponded well to patterns of female host species and host age preference for oviposition and parasitoid larval performance. In contrast, percentages of encapsulation of C. rubecula were not consistent with host preference and host suitability. We argue that encapsulation alone is unlikely to provide a sufficient explanation for C. glomerata and C. rubecula host range.     

共生菌Asaia在四种鳞翅目害虫中的感染情况

【目的】Asaia属醋酸菌已被证明与双翅目、膜翅目、半翅目和鳞翅目的多种昆虫存在着重要的共生关系。本研究检测斜纹夜蛾Prodenia litura(Fabricius)、粘虫Mythimna separata Walker、小地老虎Agrotis ypsilon Rottemberg和甘蓝夜蛾Mamestra brassicae Linnaeus体内Asaia的感染情况,并研究了Asaia的传递效率。【方法】基于16 s r DNA特异序列检测,分析Asaia在4种鳞翅目害虫中的感染情况。通过建立感染Asaia的斜纹夜蛾品系,检测其垂直传递效率。【结果】Asaia在斜纹夜蛾、粘虫、小地老虎和甘蓝夜蛾田间种群中均有感染,其感染率分别为45.8%、66.7%、47.2%和31.0%。系统发育分析表明,Asaia 16S r DNA序列可分为3种序列型ST1、ST2和ST3,其中4种昆虫中均含有ST1序列型。父母亲本均感染Asaia的斜纹夜蛾后代,Asaia感染率达到91.7%。【结论】Asaia在鳞翅目害虫中存在较高的感染率,在昆虫物种间可能存在水平传递并且在种内可进行垂直传递。     

Cellular immune responses and FAD-glucose dehydrogenase activity of Mamestra brassicae (Lepidoptera: Noctuidae) challenged with three species of entomopathogenic fungi

Abstract.  Cellular immune responses and glucose dehydrogenase activity are examined in the larvae of Mamestra brassicae (Lepidoptera: Noctuidae) injected with three entomopathogenic fungi, Beauveria bassiana , Nomuraea rileyi and Paecilomyces tenuipes . Total haemocyte count and the number of spherulocytes increase significantly over 2 h in larvae infected with N. rileyi , the most virulent fungal pathogen of M. brassicae . However, glucose dehydrogenase is not activated in the haemolymph of larvae inoculated with N. rileyi . By contrast, P. tenuipes , the least virulent fungal species, but with the highest proteolytic activity, activates glucose dehydrogenase and the number of nodules formed is significantly larger in larvae inoculated with P. tenuipes . It is suggested that the different virulence of each fungal species is caused by specific immune responses in the larvae. Haemopoiesis is affected by the fungal conidia and this is investigated by culture of the haemopoietic organs of M. brassicae in vitro . The proportion of spherulocytes discharged from the organs is typically high after all of the fungal treatments. In addition, the anterior and posterior haemopoietic organs, according to the histological locations in larva, show different patterns of haemopoiesis in response to fungi.     

Putrescine--an inhibitor of the reaction of carbon dioxide hydration in hemolymph of lepidoptera

Using high-voltage electrophoresis, thin-layer chromatography and chemical analysis tetramethylene diamine was found in hemolymph of 8 Lepidoptera species (Bombyx mori L., Ocneria dispar L., Euproctis chrysorrhoea L., Yponomeuta malinella L., Pieris brassicae L., Aporia crataegi L., Melacosoma neustria L., Mamestra brassicae L.). Its content reaches 11 mM 1 l of hemolymph. The absence of tetramethylene diamine in the intestine wall and Malpighian vessels and its presence in the fat body permits suggesting its synthesis in the fat body. Ion-selective H-electrodes were used to show the function of tetramethylene diamine as an inhibitor of the CO2 hydration reaction in the insects hemolymph.     

Effects of Pieris host species on life history parameters in a solitary specialist and gregarious generalist parasitoid (Cotesia species)

Host specificity and host selection by insect parasitoids are hypothesized to be correlated with suitability of the hosts for parasitoid development. The present study investigates the correlation between host suitability and earlier studied host-finding behaviour of two closely related braconid larval parasitoid species, the generalist Cotesia glomerata (L.) and the specialist C. rubecula (Marshall) (Hymenoptera: Braconidae). We compared the capability of both parasitoid species to parasitize and develop in three Pieris host species, i.e. P. brassicae (L.), P. rapae (L.) and P. napi (L.) (Lepidoptera: Pieridae). In laboratory experiments, we measured the effect of host species on fitness parameters such as survival, development, sex ratio and size of parasitoid progeny. The results show that C. glomerata is capable of developing in the three host species, with significant differences in parasitoid survival, clutch size and adult weight among Pieris species. The host range for development was more restricted for C. rubecula. Although C. rubecula is physiologically able to develop in P. brassicae larvae, parasitoid fitness is negatively affected by this host species, compared to its most regular host, P. rapae. A comparison of the present data on host suitability with earlier studies on host-searching behaviour suggests that the host-foraging behaviour of both parasitoid species not only leads to selection of the most suitable host species for parasitoid development, but also plays a significant role in shaping parasitoid host range.     

The host range of Plasmodiophora brassicae and its relationship to endogenous glucosinolate content

The host range of the soilborne obligate biotroph, Plasmodiophora brassicae was investigated. Evidence is presented that infection by P. brassicae might occur in non- Brassica species, leading to the potential formation of resting spores. Structures resembling P. brassicae were found in the root cortex of Tropaeolum majus , Carica papaya , Reseda alba and Beta vulgaris as demonstrated by scanning electron microscopy. Inoculation of Brassica rapa roots with spores extracted from either T. majus or B. vulgaris roots which had been previously inoculated with P. brassicae led to development of clubroot in the roots of B. rapa . It was also shown that the development of the symptom might be correlated with glucosinolate content, although other host factors are implicated in the B. vulgaris interaction with P. brassicae . In the glucosinolate-containing non-Brassicas, T. majus and C. papaya , the concentrations of benzylglucosinolate increased markedly in roots inoculated with P. brassicae , compared with the controls. There were also increases in concentrations of benzylglucosinolate in leaves of T. majus after P. brassicae infection. However, in R. alba roots, the total glucosinolate content decreased after inoculation with P. brassicae compared with the controls. High root concentrations of 2-OH-2-phenylethylglucosinolate (glucobarbarin) compared with low root indole glucosinolates in this species might limit P. brassicae infection and development. The importance of our investigations in relation to cultivation of non- Brassica species on fields infested with P. brassicae is discussed.     

The mutation R(423)S in the Bacillus thuringiensis hybrid toxin CryAAC slightly increases toxicity for Mamestra brassicae L

Bacillus thuringiensis Cry1Ac toxin is 100 times less toxic than Cry1C to Mamestra brassicae. An R(423)S mutation abolishes Cry1Ac toxin proteolysis in M. brassicae gut juice but does not increase its toxicity to this insect. The CryAAC hybrid toxin (1Ac/1Ac/1Ca) is toxic to M. brassicae but is susceptible to gut protease digestion at the R(423) residue. Accordingly we have investigated the effect of the R(423)S mutation in CryAAC on its toxicity for M. brassicae and Pieris brassicae. Bioassays demonstrated that the R(423)S mutation slightly increased the toxicity of CryAAC for M. brassicae by having a significantly inhibitory effect on the growth of surviving larvae. The mutant hybrid was still highly toxic to P. brassicae. Features of CryAACR(423)S such as, (1) stability in M. brassicae gut juice and (2) crystal solubility were investigated. Computer simulations suggest that a possible major increase in flexibility in the CryAAC loop beta7/beta8 (G(391)-P(397)) caused by the R(423)S substitution could be a reason for the increase in M. brassicae toxicity.     

Methyl salicylate, identified as primary odorant of a specific receptor neuron type, inhibits oviposition by the moth Mamestra brassicae L. (Lepidoptera, noctuidae)

The cabbage moth, Mamestra brassicae L. (Lepidoptera, Noctuidae), is a polyphagous species that is often choosing plants of Brassica as hosts for oviposition. In the search for biologically relevant odorants used by these moths, gas chromatography linked to electrophysiological recordings from single receptor neurons (RNs) has been employed, resulting in classification of distinct types of neurons. This study presents specific olfactory RNs responding to methyl salicylate (MeS) as primary odorant and showing a weak response to methyl benzoate, the 2 aromatic compounds occurring together in several plant species. In 2 cases, the neuron was colocated with another RN type responding to 6 green leaf volatiles: 1-hexanol, (3Z)-hexen-1-ol, (2E)-hexen-1-ol, (3Z)-hexenyl acetate, (2Z)-hexen-1-ol, and an unidentified compound. Whereas the specific RNs detected the minor amounts of MeS in some plants, the compound was not found by gas chromatography linked to mass spectrometry in intact plants, but it was found after herbivore attack. The behavioral effect of MeS was studied in outdoor test arenas with Brassica napus and artificial plants. These experiments indicated that mated M. brassicae females avoid plants with dispensers emitting MeS. As it is induced by caterpillar feeding, this compound may mediate a message to mated M. brassicae females that the plant is already occupied.     

Effects of Bt transgenic Chinese cabbage on the herbivore Mamestra brassicae (Lepidoptera: Noctuidae) and its parasitoid Microplitis mediator (Hymenoptera: Braconidae)

We investigated the effects of a diamondback moth-resistant Chinese cabbage (Brassica campestris subsp napus variety pekinensis Makino), expressing the insecticidal protein CrylA(c) toxin derived from Bacillus thuringiensis, on the nontarget herbivore Mamestra brassicae (L.) (Lepidoptera: Noctuidae) and its parasitoid wasp Microplitis mediator (Haliday) (Hymenoptera: Braconidae). A decreased survival rate at neonate stage was observed in M. brassicae when reared on Bt cabbage, although overall development was not significantly affected. According to enzyme-linked immunosorbent assay test using CrylA(c) antibody, the Cry toxin was only detected in the alimentary canal, not in the hemolymph or remaining body parts of M. brassicae, indicating that the ingested Cry toxin is neither distributed inside the body nor transferred through the trophic level. As expected, no Cry toxin was found in the larvae and cocoons of M. mediator. In addition, no significant changes were observed in the parasitization rate, larval period, pupal period, cocoon weight, or adult emergence rate when M. mediator wasps were reared on the M. brassicae larvae fed with transgenic Chinese cabbage. In summary, no direct or indirect adverse effects of transgenic Chinese cabbage on the two nontarget insect species were observed, suggestive of low risk in herbivore-parasitoid food chain.     

Influence of various host plants on the consumption and utilization of food by Pieris brassicae (Linn.)

Pieris brassicae (Linn.) is a destructive cosmopolitan pest of cruciferous crops. It is present wherever its host plants occur, and it is considered to be one of the most widely distributed of all the Lepidoptera. We investigated the affect of various host plants on the food consumption and utilization by P. brassicae. We quantified consumption of food, larval duration, pupal duration and weight on cabbage (Brassica oleracea var. capitata), cauliflower (Brassica oleracea var. botrytis), radish (Raphanus sativus), broccoli (Brassica oleracea var. italica) and mustard (Brassica campestris) under laboratory conditions. Insect-host relationships can be better understood by knowing the rate of food consumption, its digestibility and conversion of food eaten to body tissue. The consumption of food generally increased with the advancement of larval age. In our study we found that consumption of food was highest on radish and lowest on broccoli. The highest consumption of a particular host does not always indicate greater suitability of that host, until and unless other factors like consumption index (CI), relative growth rate (RGR), efficiency of conversion of ingested food (ECI), approximate digestibility (AD) and efficiency of conversion of digested food (ECD) are also considered. In the current investigation, factors like CI, RGR, ECI and ECD were highest on cabbage. Low body weight of pupa is associated with rapid development. On cabbage, the weight of pupa of both sexes was found lowest. Thus, from the present study, it can be concluded that cabbage is a more suitable host for P. brassicae than other host plants evaluated. Hence, on cabbage, the values of Waldbauer indices were highest and P. brassicae developed with a faster rate.     

The presence of carotenoids in various species of Lepidoptera

The presence of 41 carotenoids in 114 species of Lepidoptera was determined. The carotenoids characteristic of butterflies were zeaxanthin, β-cryptoxanthin, lutein epoxide, astaxanthin, lycopene, torulene and canthaxanthin.     

Nutritional suitability of individual carbohydrates and amino acids for adult Pieris brassicae

Abstract Many adult Lepidoptera are dependent on carbohydrate-rich solutions such as nectar and honeydew. These food sources can contain a range of carbohydrates as well as low levels of other compounds, including free amino acids, proteins and lipids. Little is known about the nutritional value of individual nectar sugars and amino acids for adult Lepidoptera. Ten sugars occurring in nectar and honeydew were tested with respect to their effects on a number of Pieris brassicae life-table parameters. In similar experiments it was investigated whether the butterfly derives nutritional benefits from mixtures of common non-essential or essential amino acids when provided either in water or in a sucrose solution. Food sources were found to have a significant effect on butterfly fertility, longevity, lifetime fecundity and daily oviposition rate. Whereas a number of sugars significantly increase longevity, melibiose and melezitose reduce P. brassicae longevity. Butterfly fecundity is compromized significantly by sucrose and melibiose. Glucose is the only sugar that has a positive effect on both longevity and fecundity as well as a number of oviposition parameters. Amino acids seem to have no or only a minor effect on the life-table parameters tested. However, when provided in sucrose, they appear to compensate for the negative effect of pure sucrose on fecundity. A comparison with an earlier study on the gustatory response of P. brassicae shows that there is only a weak correlation between the nutritional value of sugars and amino acid mixtures and their perceptibility.     

Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac delta-endotoxin

Bacillus thuringiensis protein delta-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the B. thuringiensis Cry1Ac delta-endotoxin towards Pieris brassicae (family: Pieridae) and Mamestra brassicae (family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against P. brassicae than against M. brassicae larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from P. brassicae, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of approximately 56 kDa, whereas proteases from M. brassicae, the non-susceptible insect, generated products with molecular masses of approximately 58, approximately 40, and approximately 20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M. brassicae proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by M. brassicae proteases. However, this mutation had little effect on toxicity to M. brassicae. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.     

Using COI gene sequence to barcode two morphologically alike species: the cotton bollworm and the oriental tobacco budworm (Lepidoptera: Noctuidae)

Due to limited morphological difference, the two closely related sister species, the cotton bollworm, Helicoverpa armigera (Hübner) and the oriental tobacco budworm, H. assulta (Guenée) (Lepidoptera: Noctuidae), are very difficult to distinguish, especially at the larvae stage. Recently, DNA sequence has been widely used as a bio-barcode for species identification. In this study, we attempted to distinguish H. armigera and H. assulta using the mitochondrial cytochrome C oxidase subunit I gene (COI) gene sequence as the barcode. We determined a 658 bp segment of the COI gene for 28 individuals of H. armigera, 8 individuals of H. assulta, and 10 individuals of Mamestra brassicae (as the outgroup) in Yunnan Province, southwest of P. R. China, together with one H. assulta and two H. armigera reported sequences from GenBank. Twenty-three haplotypes were identified in all 49 samples. As expected, network analysis of the haplotypes of the three species presented a clustering pattern consistent with the respective species status. Haplotypes of the same species differed from each other by no more than three nucleotide substitutions. However, each haplotype of H. armigera differed from that of H. assulta by at least 22 nucleotide substitutions. Both species differed from M. brassicae by more than 50 nucleotide substitutions. 17 unique diagnostic nucleotides were also used to discriminate the two species. The finding of large COI sequence differences between H. armigera and H. assulta suggested that it could be used to distinguish the two morphologically alike species and be employed for quick species identification during pest control.     

Superparasitism in Cotesia glomerata: response of hosts and consequences for parasitoids

Abstract.  1. Superparasitism occurs in Cotesia glomerata (Hymenoptera: Braconidae), a gregarious endoparasitoid of Pieris spp. (Lepidoptera: Pieridae). The response of P. brassicae larvae to superparasitism and the consequences for the parasitoid were examined in order to elucidate the ecological significance of this behaviour.
2. Field surveys of a Swiss population revealed that C. glomerata brood sizes from P. brassicae larvae ranged from three to 158, and both the female ratio and the body weight of emergent wasps correlated negatively with brood size. In the laboratory, single oviposition on P. brassicae larvae did not produce any brood size larger than 62, but brood size increased with superparasitism.
3. Laboratory experiments demonstrated that both naive and experienced female wasps were willing to attack hosts that had been newly parasitised by themselves or conspecifics. Superparasitism reduced survivorship but increased food consumption and weight growth in P. brassicae larvae. Superparasitism lengthened parasitoid development and prolonged the feeding period of host larvae.
4. Despite a trade-off between maximising brood size and optimising the fitness of individual offspring, two or three ovipositions on P. brassicae larvae resulted in a greater dry female mass than did a single oviposition on the host. Thus, superparasitism might be of adaptive significance under certain circumstances, especially when host density is low and unparasitised hosts are rare in a habitat.