Characterization of three serotonin receptors from the small white butterfly,Pieris rapae

Serotonin (5-hydroxytryptamine, 5-HT) plays a key role in modulating diverse physiological processes and behaviors in both protostomes and deuterostomes. These functions are mediated through the binding of serotonin to its receptors, which are recognized as potential insecticide targets. We investigated the sequence, pharmacology and tissue distribution of three 5-HT receptors (Piera5-HT_1A, Piera5-HT_1B, Piera5-HT₇) from the small white butterfly Pieris rapae, an important pest of cultivated cabbages and other mustard family crops. Activation of Piera5-HT_1A or Piera5-HT_1B by 5-HT inhibited the production of cAMP in a dose-dependent manner. Stimulation of Piera5-HT₇ with 5-HT increased cAMP level significantly. Surprisingly, with the exception of 5-methoxytryptamine, agonists including α-methylserotonin, 8-Hydroxy-DPAT and 5-carboxamidotryptamine activated these receptors poorly. The results are consistent with previous findings in Manduca sexta. All three receptors were blocked by methiothepin, but ketanserin and yohimbine were not effective. The selective mammalian 5-HT receptor antagonists SB 216641 and SB 269970 displayed potent inhibition effects on Piera5-HT_1B and Piera5-HT₇ respectively. The results we achieved here indicate that the pharmacological properties of Lepidoptera 5-HT receptors are quite different from those in other insects and vertebrates and may contribute to development of new selective pesticides. This study offers important information on three 5-HT receptors from P. rapae that will facilitate further analysis of the functions of 5-HT receptors in insects.     

The effect of host acceptability on oviposition and egg accumulation by the small white butterfly, Pieris rapae

The influence of host‐plant acceptability on oviposition rate, egg load, internal fat storage and longevity was studied in the small white butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Newly emerged females and males were presented with either cabbage (Brassica oleracea L. var. gemmifera), which is a highly acceptable host for the small white, or wallflower (Cheiranthus cheiri L.), which is much less acceptable for oviposition. Individuals were dissected when 3, 6 and 9‐days‐old. Females kept on cabbage contained fewer mature eggs than those kept on wallflower and there was no change in the load of mature eggs over the time course studied. The number of immature eggs carried did not vary with host plant species, but did fall significantly between days 6 and 9. The body fat content of individuals declined with increasing age, but the decline was slower for individuals kept on the host of low acceptability. Individuals that were allowed to spend their natural lifespan on cabbage showed similar oviposition patterns over time, where the oviposition rate started high and gradually reduced until death. However, considerable differences in oviposition pattern were found in individuals kept on wallflower, varying from that found on cabbage to no oviposition at all. The implications of these findings are discussed in the light of existing oviposition theories. This leads to the conclusion that in one species the premises of existing theories on optimization of oviposition are not mutually exclusive but rather play in concert.     

Avoidance of maladaptive,precocious copulation in the cabbage white butterfly,Pieris rapae crucivora

In the cabbage white butterfly, Pieris rapae crucivora, copulation hinders normal expansion and hardening of the wings of newly emerged females. The resulting permanent wing deformation makes it impossible for females to fly and therefore, to find an oviposition site and nectar sources. An attempt was made to clarify whether the newly emerged female butterfly avoids copulation. Observation of wing expansion and hardening reveals that the wings are fully expanded and hard by 20–30 min after emergence. In the field, presentation of females with soft wings to males shows that males will attempt to copulate with these females. However, newly emerged females prevent successful completion of copulation by assuming the mate refusal posture, and thereby avoid a potentially maladaptive copulation. The discussion focusses on the question as to why females and not males avoid early copulation.     

The learning abilities of the white cabbage butterfly,Pieris rapae,foraging for flowers

This study examines the role of learning and memory in the butterflyPieris rapae crucivora Boisduval during foraging for flowers. In an outdoor cage with 6 flower species,P. rapae showed various visiting patterns: some visited only one species, while others visited several species in a day. The foraging process for flowers ofErigeron annuus (L.) Pers. could be divided into two successive steps: (1) landing on the nectaring caputs, and (2) finding the source of nectar in the caput. Butterflies learned to proceed through the two steps more efficiently with successive attempts: they gradually decreased landings on nectarless caputs and probings on the nectarless petals of ligulate flowers respectively. As a result, handling time per unit caputs became shorter, and apparent rewards per unit time, i.e. the efficiency of collecting nectar, increased. In addition, once learned,P. rapae could remember a rewarding flower color for 3 days, which was not interfered with by learning another flower color. This indicates thatP. rapae keeps memory for a period longer than 3 days, and that they can remember at least two flower species as suitable flower resources. Furthermore, data indicated that they sometimes can apply the foraging skills obtained on other flower species to a novel one. These abilities could enable butterflies to easily switch flower species, or to enhance labile preference. It has been known thatP. rapae also shows flower constancy, which may be due to memory constraints. Therefore, they may appropriately use two foraging tactics: visit consistency and labile preference, to get enough nectar according to their circumstances.     

A review of the pre-oviposition behaviour of small cabbage white butterfly, Pieris rapae(lepidoptera: Pieridae)

This paper reviews the literature concerning the ovipositional behaviour of Pieris rapae (small cabbage white butterfly) and where appropriate considers results from studies involving other butterflies. The paper considers searching behaviour, stimuli utilised in host plant finding and identification and concludes with a general section on the role of learning and prior experience in ovipositional behaviour.     

Female size and age-specific fecundity in the small white butterfly,Pieris rapae crucivora Boisduval (Lepidoptera: Pieridae)

Individual differences in several reproductive parameters of female Pieris rapae were investigated in a controlled laboratory condition. Lifetime and age-specific fecundity showed considerable variability between individuals. Larger females began oviposition at an earlier age than smaller ones, and larger females were more fecund than smaller ones. Larger females laid a larger proportion of their eggs in the early stages of their reproductive lifetime, whilst smaller females laid the larger proportion of their eggs later in their reproductive lifetime. The significance of the variance in age-specific fecundity associated with female size is discussed with respect to the seasonal change in size and habitat utilization of this species.     

Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A‐dependent manner in non‐neuronal and neuronal cell lines

Trafficking of G protein‐coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein‐coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non‐neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand‐mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand‐mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A‐dependent manner.

     

Comparative analysis of two biliproteins, BP1 and BP2, from haemolymph of cabbage white butterfly, Pieris rapae

Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs.     

Transient expression of serotonin 5‐HT4 receptors in the mouse developing thalamocortical projections

The serotonin 5‐HT₄ receptor (5‐HT₄‐R) is an unusually complex G‐protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5‐HT₄‐Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT‐PCR. The developing thalamocortical projections transiently expressed 5‐HT₄‐Rs in the embryonic brain and the 5‐HT₄‐R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13–17, the forebrain mRNA levels of the 5‐HT_4(a)‐R and 5‐HT_4(b)‐R splice variants increased nine‐ and fivefold, respectively, whereas the levels of the 5‐HT_4(e)‐R and 5‐HT_4(f)‐R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5‐HT₄‐R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010.     

Role of glycosphingolipids in the function of human serotonin1A receptors

Glycosphingolipids are essential components of eukaryotic cell membranes and are involved in the regulation of cell growth, differentiation, and neoplastic transformation. In this work, we have modulated glycosphingolipid levels in CHO cells stably expressing the human serotonin_1A receptor by inhibiting the activity of glucosylceramide synthase using (±)‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), a commonly used inhibitor of the enzyme. Serotonin_1A receptors belong to the family of G‐protein‐coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral, and developmental functions. We explored the function of the serotonin_1A receptor under glycosphingolipid‐depleted condition by monitoring ligand‐binding activity and G‐protein coupling of the receptor. Our results show that ligand binding of the receptor is impaired under these conditions although the efficiency of G‐protein coupling remains unaltered. The expression of the receptor at the cell membrane appears to be reduced. Interestingly, our results show that the effect of glycosphingolipids on ligand binding caused by metabolic depletion of these lipids is reversible. These novel results demonstrate that glycosphingolipids are necessary for the function of the serotonin_1A receptor. We discuss possible mechanisms of specific interaction of glycosphingolipids with the serotonin_1A receptor that could involve the proposed ‘sphingolipid‐binding domain’.     

Fine structure of the stretch receptor in the bursa copulatrix of the butterfly,Pieris rapae crucivora

Summary A pair of multipolar stretch-receptive neurons were found in the bursa copulatrix of the female cabbage white butterfly, Pieris rapae crucivora. The cell body of each neuron, about 10 m in diameter, lies on the edge of the muscular region in the antero-lateral wall of the corpus bursae. No special accessory structure, such as a receptor muscle, is associated with the neuron. The several dendrites extend radially into the muscle layer. The dendrites are ensheathed except for their terminal tips, and, on their course, they anchor repeatedly on the epithelial cells or the muscle fibers in such a manner that their basement membranes fuse together. While the ensheathed dendrite is usually 0.1–0.2 m in diameter, it often forms 1–2 m varicosities especially at anchor sites, so that it looks like a varicose, or beaded, chain. The varicosities contain a number of mitochondria, but only microtubules are found in the fine interconnecting parts of the dendrite. The naked dendritic tips terminate in the basement membrane of the epithelial cell. The varicosities, as well as naked tips, seem to be important for stimulus transduction in the sensory cell of this type.     

Associative learning in the ovipositional behaviour of the cabbage butterfly, Pieris rapae

ABSTRACT. When tested with familiar and novel oviposition sites, gravid female Pieris rapae (L.) tended to land on sites of the same appearance as those they had experienced 24 h earlier. This acquired preference could be reversed by subsequent e xperience. An ovipositional preference, shown by groups of insects, paralleled the landing preferences of single insects. These influences of experience were shown with intact cabbage plants of different kinds, with discs cut from the leaves of different kinds of cabbage, and with discs of blotting paper of different shades of green and blue, wetted with sinigrin solution or water. A single landing by a gravid female on paper wetted with sinigrin solution induced a landing preference for the same colour of paper in a choice test made 24 h later. Gravid females thus associated a favourable chemical stimulus of oviposition with the appearance of the acceptable site. Such learning by trial and error would facilitate host-selection from a distance. In the laboratory it induced oviposition on 'neutral' sites of the same appearance as 'host' sites.     

On the fate of seasonally plastic traits in a rainforest butterfly under relaxed selection

Many organisms display phenotypic plasticity as adaptation to seasonal environmental fluctuations. Often, such seasonal responses entails plasticity of a whole suite of morphological and life‐history traits that together contribute to the adaptive phenotypes in the alternative environments. While phenotypic plasticity in general is a well‐studied phenomenon, little is known about the evolutionary fate of plastic responses if natural selection on plasticity is relaxed. Here, we study whether the presumed ancestral seasonal plasticity of the rainforest butterfly Bicyclus sanaos (Fabricius, 1793) is still retained despite the fact that this species inhabits an environmentally stable habitat. Being exposed to an atypical range of temperatures in the laboratory revealed hidden reaction norms for several traits, including wing pattern. In contrast, reproductive body allocation has lost the plastic response. In the savannah butterfly, B. anynana (Butler, 1879), these traits show strong developmental plasticity as an adaptation to the contrasting environments of its seasonal habitat and they are coordinated via a common developmental hormonal system. Our results for B. sanaos indicate that such integration of plastic traits – as a result of past selection on expressing a coordinated environmental response – can be broken when the optimal reaction norms for those traits diverge in a new environment.     

Profiling two indole‐2‐carboxamides for allosteric modulation of the CB1 receptor

Allosteric modulation of G‐protein coupled receptors (GPCRs) represents a novel approach for fine‐tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole‐2‐carboxamides:5‐chloro‐3‐ethyl‐1‐methyl‐N‐(4‐(piperidin‐1‐yl)phenethyl)‐1H‐indole‐2‐carboxamide (ICAM‐a) and 5‐chloro‐3‐pentyl‐N‐(4‐(piperidin‐1‐yl)phenethyl)‐1H‐indole‐2‐carboxamide (ICAM‐b). Although both ICAM‐a and ICAM‐b enhanced CP55, 940 binding, ICAM‐b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G‐protein coupling to CB1, ICAM‐b induced β‐arrestin‐mediated downstream activation of extracellular signal‐regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.     

Pattern of spatial distribution of the common cabbage butterfly,Pieris rapae in a cabbage farm

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Contributions from the Entomological Laboratory, Kyoto University, No. 203.     

拟青霉菌代谢产物对菜粉蝶幼虫的生物活性

淡紫拟青霉(Paecilomyces lilacinus)甲醇萃取物柱层析活性馏分DM6和拟青霉(Paeecilomyces sp.)菌株X1甲醇萃取物柱层析活性馏分X1M5对菜粉蝶Pieris rapae(L.)4龄幼虫具有较强的选择性和非选择性拒食作用,处理后24h选择性AFC50分别为5.8652mg/mL和3.5239mg/mL,非选择性AFC50分别为9.0095mg/mL和14.0234mg/mL。饲喂法、注射法、点滴法测定结果表明,DM6和X1M5对菜粉蝶4龄幼虫具有较强的毒杀活性,其中注射法处理后72h,DM6和X1M5对菜粉蝶4龄幼虫的LD50分别为0.1846μg/头和0.6784μg/头。DM6和X1M5以5.0mg/mL饲喂法处理菜粉蝶3龄幼虫后,试虫生长发育受到强烈的抑制作用,发育抑制率分别为73.34%和63.30%。这些结果对于进一步研究拟青霉菌杀虫活性成分具有重要参考意义。