Haemopoietic organs and effect of their ablation on total haemocyte count in lemon-butterfly,Papilio demoleus L

The haemopoietic organs in Vth instar larvae of P. demoleus are in the form of thin transparent cellular sheets, closely wrapped around the base of each wing-pad. Three cell types viz; prohaemocytes, plasmatocytes and oenocytoids appear to be derived from these organs and their ablation caused a reduction in cell number which, in turn, revealed that the haemocytes in general are derived both from the haemopoietic organs as well as from the circulating blood cells.     

Neuroendocrine organs of the lemon-butterfly, Papilio demoleus L. I. The corpora cardiaca-allata complex of the larva.

The histomorphology of the retrocerebral endocrine organs of the larva of the lemon-butterfly, P. demoleus has been described employing a conventional neurosecretory staining technique. The larval organs lie a little away from the brain and oesophagus and, therefore, are not sub-aortic in position unlike most other insects and the adult of this species itself. There are two long NCCs which innervate other target organs in addition to the CC. The recurrent nerve of the stomatogastric nervous system retains its normal connections with the CC even in absence of the hypocerebral ganglion. Histological evidence suggests that the NSM inside the CC remains intraaxonal without being possibly unloaded in the substance of these organs. Axonal transport of NSM to target organs is also in evidence and though NSM is seen in the NCA, it could not be detected inside the CA.     

Neuroendocrine organs of the lemon-butterfly, Papilio demoleus L. II. The corpora cardiaca-allata complex of the adult.

The retrocerebral endocrine organs of the adult lemon-butterfly; Papilio demoleus have been described. The organs are subaortic lying closely behind the brain. While the nervi corporis cardiaci I (NCCI) originate from the protocerebrum of the brain, the NCCII seem to take their origin in the tritocerebrum in common with another nerve named earlier as the tegumentary nerve. The corpora cardiaca (CC) and corpora allata (CA) are closely approximated to each other obliterating the nervi corporis allati (NCA) which are conspicuous in the larva of the same species. An intercardiacal bridge (ICB) connects the CC of the two sides and acts as a possible centre of distribution for the neurosecretory material (NSM) to the gut. Histological evidence suggests that the NSM inside the CC remains intraaxonal without being primarily unloaded in the organs for storage. The intrinsic secretory cell of the CC are intimately associated with the neurosecretory fibres from the brain and bear fairly thick axons. No NSM could be detected in the CA of this insect.     

广州地区达摩凤蝶生物学特性观察

详细描述了达摩凤蝶Papilio demoleus L．各虫态的形态特征、生活史及生活习性。在广州地区。该蝶1年发生5代以上,以蛹在寄主植物上越冬,成虫3月初始见,幼虫共5龄,以芸香科的金橘属、酒饼筋和柑橘类等植物叶片为食料。     

Preservatives influence induction of feeding tolerance in Papilio demoleus larvae

The feeding responses of newly hatched larvae of Papilio demoleus on the diet with preservatives were as high as on the diet without preservatives though less than those on the host lime leaves. However, when the larvae were reared upto the 5th instar on the host lime leaves or the diet with or without preservatives, their feeding responses towards host lime leaves and the diet without preservatives were equally high. The diet with preservatives reared larvae showed 100% response when tested on the same diet. But their response towards diet with preservatives declined to 80% when the larvae were reared on the diet without preservatives and to 10% when they were reared on the host lime leaves. The larvae seem to have an aversion against the preservatives if reared in their absence which was enhanced by their experience of feeding on the host leaves during their ontogeny.

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Influence des agents de conservation sur la prise de nourriture par les chenilles de Papilio demoleus

Résumé La prise de nourriture par des chenilles néonates de P. demoleus est aussi élevée sur régime avec agents de conservation qu'en leur absence, bien qu'elle soit inférieure à celle qui est observée sur les feuilles de son hôte Citrus limettioides. Cependant, quand les chenilles ont été élevées jusqu'au 5ème stade sur des feuilles de C. limettioides, ou sur des régimes avec ou sans conservateurs, leur prise de nourriture sur feuilles de C. limettioides, et régime sans conservateur est aussi importante. Toutes les chenilles élevées sur régime avec conservateur ont consommé lorsqu'elles avaient été maintenues sur le même régime. Par contre, leurs réactions face à un régime contenant des conservateurs a entraîné une diminution de près de 20% après élevage sur un régime sans conservateur, et de près de 90% après élevage sur feuilles de C. limettioides. Les chenilles semblent manifester de l'aversion envers les conservteurs si elles ont été élevées en leur absence, aversion renforcée lorsqu'il y avait eu préalablement alimentation sur feuilles de C. limettioides.

     

Host-plant relations of the citrus butterfly Papilio demoleus L.: Orientational and ovipositional responses

The selection of a suitable plant for the establishment of Papilio demoleus is done by its females and involves their two main behavioural responses: (1) Orientation and (2) Oviposition, involving abdominal-curling followed by egg-delivery. The ovipositing females visit the host (Citrus) and non-host plants almost equally due to their almost equally high attraction to the green or yellowish-green colour of both the plants. On perceiving the specific attractant odour emitted by the ether-soluble constituents of the host plant (Citrus), the attraction of the butterflies to these plants becomes greater than that to the non-host plants and increases the chances of their visiting the host plants. Subsequently, the contact chemical stimuli provided by the ether as well as 80% ethanol-soluble constituent of the host plants, in combination with their odour and colour, elicit the ovipositional response of the butterflies. If the odour and contact stimuli are not appropriate, as in the case of non-host plants, the butterflies depart and visit other plants until they come to a host plant providing the above-mentioned combination of visual, olfactory and contact chemical stimuli to elicit increased attraction and ovipositional response.

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Résumé Ce travail étudie de rôle des réponses comportementales d'orientation et de ponte de Papilio demoleus, comme éléments déterminant sa sélection et son installation à l'égard d'une série de plantes: la plante-hôte principale Citrus limettioidés, l'hôte occasionnel Aegle marmelos et des plantes non-hôtes, Coriandrum sativum, Rosa indica, Gossypium hirsutum.Les réponses d'orientation (attraction) et de ponte du papillon à l'egard de la plante-hôte principale (Citrus) s'accroissent avec l'âge d'insecte, atteignant un maximum chez les femelles âgées de 3–4 jours, puis diminuent. Les papillons montrent une attraction presque aussi forte pour les plantes-hôtes et les plantes non-hôtes, mais les réponses consistant dans le recourbement de l'abdoment et le dépôt d'oeufs sont fortes pour Citrus, moindres par A. marmelos et nulles pour les plantes non-hôtes.Les stimuli visuels des plantes-hôtes et non-hôtes, en particulier la coloration vert ou jaune-vert, déclenchent une forte attraction mais non les réponses liées à la ponte. Ceci est confirmé par des tests avec des colorants de diverses couleurs. L'humidité ne provoque aucune réponse des papillons.Un extrait éthéré des feuilles de Citrus attire les papillons quand celui-ci est présenté sur un tissu de mousseline imprégné d'eau; il déclenche également les réflexes de ponte. Un extrait dans l'éthanol 80%, présenté sur un tissue humide ne provoque qu'une attraction faible et aucune réponse de ponte. Les stimuli de contact que l'on peut provoquer avec la fraction soluble dans l'éther et la fraction soluble dans l'éthanol 80% entrainent de fortes réactions de la courbure abdominale et de ponte. La combinaison d'une couleur vert jaunâtre, d'eau, d'odeur de l'extrait éthéré et de stimuli de contact avec des extraits dans l'éther et l'éthanol, déclenche les trois types de réponse des papillons.

     

Effect of repeated sampling on haemolymph pH,PO2 and haemocyte activity in the Pacific oyster,Crassostrea gigas (Thunberg)

The effects of repeat bleeding via a novel cannulation technique, on the physiology of the Pacific oyster, Crassostrea gigas (Thunberg), were compared to control animals from which haemolymph was sampled by syringe and needle. The effects of surgery and cannulation were characterized by an initial alkalosis (up to 8 h) in the haemolymph of cannulated animals followed by an acidosis of up to 12 h; which occurred in both cannulated and control oysters. A decline in partial pressure of oxygen of oyster haemolymph, observed in both cannulated and control oysters, was directly related to shell closure following handling. Furthermore, a haemocyte concentration observed at 4 h followed by haemocyte dilution at 12 h post-surgery, suggested a handling-related stress response in both cannulated and control oysters. A heightened phagocytic activity of haemocytes at 4 h also supported the occurrence of handling-related stress response in all animals. The cannulation technique described here provides the investigator with a convenient tool for chronic, and repeated sampling of haemolymph with a transient disturbance to the animal.     

Relationship between the olfactory sensilla of Papilio demoleus L. larvae and their orientation responses to different odours

Papilio demoleus larvae showed maximum attraction to odours of citral, cirtonellal and citronellol, slightly less to geraniol and 1-limonene, and low attraction to hydroxy-citronellal and geranyl acetate. These chemicals (except the last) are the main odorous constituents of the insect's host plant (Citrus) leaves. Phellandrene was almost inert, whereas farnesol and d-limonene elicited a low, and linalool, eugenol and clove oil a high repulsion. Larvae deprived of one antenna and both maxillary palps were similar to normal larvae in their orientational responses. Normal larval responses changed with the inactivation of one or more of the three specific antennal sensilla. A full complement of the three sensilla was not essential for the normal larval response to certain chemicals. Any of the sensilla singly or in combination with any other sensillum could mediate the normal larval response (attraction) to citral and geraniol. For a few other chemicals, certain specific sensilla, singly or in a combination of two, mediated the normal response. Only for d-limonene were all three sensilla essential for the normal response.     

Structure and metamorphic changes in the brain of the lemon butterfly Papilio demoleus L. (Lepidoptera: Papilionidae)

The morphology of the larval and adult brain of Papilio demoleus, and changes in the cell population and neuropile morphology during the pupal period have been described. The larval brain has more simple fibre areas than that of the adult. Dividing neuroblasts have been found which form the adult neurones. The larval brain contains the three neuromeres (proto-, deuto-, and tritocerebrum). The protocerebrum has well developed corpora pedunculata, a central body, a pons cerebralis and developing optic centres. The corpora ventralia are joined with each other by paired ventral commissures (single in adult). The deutocerebrum is simple and small, the antennal centres are small and simple (ef. adult). The glomerular tritocerebrum is posteroventral to the deutocerebrum, and fibres from the former travel to the crura cerebri. The cortex of the brain consists of four types of glial cells and of association cells, and large and medium sized motor neurones. The number of mitoses is greatest in the larval and prepupal stages; in the pupa it decreases gradually and in late stages it does not occur. Histolysis and pyknosis begin in the prepupa and decrease considerably in the late pupa. The entire neural lamella is broken down in the early pupa. Numerous haemocytes penetrate the laminae of the neural lambella and envelop the entire brain. In the adult, behind the well-developed central body is an ellipsoid body. The medulla interna is divided into two smaller lobes and the deutocerebral lobes are differentiated into cortical and medullary zones. Chiasmata between optic centres are also formed during the pupal period.     

Flow cytometric assessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, haemolymph

The morphology and functions of haemocytes from the haemolymph of the European oyster, Ostrea edulis, were analysed by flow cytometry on the basis of cellular structures and incorporation of fluorescent markers. O. edulis circulating haemocytes appear to be composed of one to three cell populations based on cell size and granularity, with many individual variations. Analysis of haemocytes after propidium iodide staining indicated that the majority of oyster haemocytes are alive after sampling. The phagocytic activity level of haemocytes was analysed using fluorescent beads and this cell activity varied greatly depending on the oysters. The use of 3,3'dihexyloxacarbocyanine iodide (DIOC6) allowed the demonstration of several cell populations on the basis of labelled intensity. One to three cell sub-populations can be observed depending on the oysters. The haemocytes characterised by high granularity showed a strong fluorescence intensity related to high mitochondrial activity.     

四个达摩凤蝶新孵幼虫细胞系的建立及其生物学特性

【目的】建立达摩凤蝶 Papilio demoleus Linnaeus新孵幼虫细胞系,并对其生物学特性进行研究。【方法】使用改良配方的Grace培养基并辅以20%胎牛血清,通过原代和传代培养建立达摩凤蝶新孵幼虫细胞系。通过显微观察、细胞活力分析、核型分析和分子鉴定,获得4个新建细胞系的生物学特性数据。使用Bac-To-Bac杆状病毒表达系统构建重组分泌型碱性磷酸酶杆状病毒(AcMNPV-SEAP)。使用有限稀释法测定达摩凤蝶细胞系对AcMNPV-SEAP的半数组织培养感染剂量(TCID₅₀), 比较达摩凤蝶细胞系对AcMNPV-SEAP的敏感性。【结果】建立了4个贴壁生长的达摩凤蝶新孵幼虫细胞系(RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3及RIRI-PaDe-4),且均传至60代以上。形态学方面,细胞系RIRI-PaDe-1和RIRI-PaDe-4较为相近,均由圆形、梭形以及多边形细胞组成,细胞系RIRI-PaDe-2主要为圆形细胞,而RIRI-PaDe-3主要为类似表皮细胞和纤维状细胞。细胞增殖动力学方面,4个细胞系的群体倍增时间分别为69.77, 67.42, 81.48及65.43 h。核型分析显示4个细胞系染色体数量均呈正态分布,其中RIRI-PaDe-2, RIRI-PaDe-3以及RIRI-PaDe-4的染色体数目分布区间比较接近,在45~97条之间,RIRI-PaDe-1的染色体条数相比偏少,在36~90条之间。通过比对4个达摩凤蝶细胞系和虫卵的细胞色素c氧化酶亚基I(COI)基因序列,证明4个新建细胞系来源于达摩凤蝶组织。比较4个细胞系对AcMNPV-SEAP敏感性发现RIRI-PaDe-3对此病毒较为敏感,可作为重组杆状病毒表达的宿主。【结论】虽然4个新建达摩凤蝶细胞系的来源相同,具有相同的遗传背景,但其生物学特征仍有明显差异,具有进一步研究的价值。     

Morphology,life cycle and management of two invasive subspecies of Papilio demoleus (Lepidoptera: Papilionidae): A review

Papilio demoleus L., also known as the citrus butterfly or chequered swallowtail, is a common species in the Asia-Pacific region, with two of its subspecies (P. d. demoleus and P. d. malayanus) posing significant threats to the citrus industry in Asia and other regions of the world. Its predominant natural host plants are the Rutaceae for the invasive northern subspecies and Fabaceae for the southern subspecies. The morphology and development of this species are reasonably well studied, although further information is required on its natural mortality rates and environmental tolerance in order to accurately predict its future potential spread throughout the Middle East and Central Americas. Synthetic pyrethroids such as deltamethrin are highly effective for the control of P. demoleus larvae. However, better control is likely to be found using an integrated approach to pest management, incorporating techniques such as biocontrol agents, microbial pesticides and phytopesticides, in conjunction with synthetic pesticides where required.     

Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.)

Sampling blood has an effect on both the number of circulating haemocytes and the concentration of copper present in the haemolymph of Eledone cirrhosa. Haemocytes were sampled over 4 h and over 7, 10 and 24 days. The number of haemocytes per millilitre significantly increases (P<0.05) within 2 h of the 4-h sample period and within 24 h of the 7-, 10- and 24-day sampling periods. Haemocyte counts, however, significantly (P<0.05) decreased within 4 h of the 4-h sample and within 3 days of the 7- and 10-day sampling periods. Over the 24-day sampling periods, haemocyte counts per millilitre decreased significantly (P<0.05) by day 5 but had significantly (P<0.05) increased again by day 17. The percentage of haemocytes with Giemsa-positive cytoplasmic granules significantly increased (P<0.05) over the 4-h sampling period and over 24 h for both the 10- and 24-day sampling period. Methods for acid phosphatase, peroxidase, protein and carbohydrate gave variable staining results over 10 and 24 days. Increased staining was observed on the second and third sampling times for both protein and acid phosphatase over the 10-day sampling period whereas increased positive results were observed for all stains over the 24-day sampling period. The concentration of copper in the haemolymph decreases within 4 h of the 4-h sampling period and continues to decrease over an 11-day sampling period. Protein values decreased over the 4-h sampling period but showed no significant change between the first and last samples over 7 days.     

Effects of decapitation at the head critical period for moulting on haemolymph ecdysteroid titres in final-instar male and female Rhodnius prolixus (Hemiptera)

Decapitation experiments performed on large populations of carefully staged last-instar Rhodnius prolixus indicate that there is a 24 h sex difference in the time of the head critical period. Males pass the head critical period over a 36 h period beginning at 1200 A.Z.T. (Arbitrary Zeitgeber Time) day 6 after feeding, while females pass the head critical period over a 36 h period beginning at 1200 A.Z.T. day 5 after feeding. Radioimmunoassay determinations of haemolymph ecdysteroid titres show that there is also a 24 h sex difference in the time of the major increase in ecdysteroid titre that commences at the head critical period. These results imply that there is a sex difference in the time at which the release of prothoracicotropic hormone (PTTH) associated with the head critical period occurs. Decapitation prior to the head critical period results in a rapid, permanent decrease in ecdysteroid titre, while decapitation after the head critical period does not prevent normal increases in ecdysteroid levels. We infer that the presence of the head is essential until just prior to the head critical period and its associated increase in at the head critical period also indicates that the head critical period represents a change in the activity of the prothoracic glands indicative of a response to a significant release of PTTH at this time. The significance of these results is discussed in relation to both classical and recent evidence concerning the timing of PTTH release and changes in ecdysteroid titres in Rhodnius prolixus.     

A novel biorational pesticide: efficacy of methionine against Heraclides (Papilio) cresphontes, a surrogate of the invasive Princeps (Papilio) demoleus (Lepidoptera: Papilionidae)

ABSTRACT The Southeast Asian citrus-feeding butterfly Princeps (Papilio) demoleus (L.) was recently introduced into the Americas, causing an imminent threat to citrus production and ornamental flora. The human nutrient amino acid methionine has been shown by us to disrupt aminoacid-modulated ion transport systems in caterpillars and other insect larvae that possess an alkaline midgut. Heraclides (Papilio) cresphontes was bioassayed as a United States Department of Agriculture permitted surrogate of the Florida quarantined P. demoleus to test the potential efficacy of methionine. Larvae were allowed to feed ad libitum on wild lime plants with leaves treated with methionine or proline. Methionine caused 100% mortality in first through fourth instars in a time- and dose-dependent manner, as determined by probit analysis whereas proline was not toxic. Wild lime host plants did not exhibit phytotoxicity with methionine treatments during a 14 d test period. It is concluded that methionine is an effective larvicide against H. cresphontes, and therefore may be a candidate environmentally safe biorational pesticide for use against invasive P. demoleus in the Americas.     

Studies on the proteins of Poecilocera picta. I-changes taking place in the haemolymph protein during moulting

The haemolymph proteins of the 6th nymph of P. picta were fractioned by the polyacrylamide gel disc electrophoresis. A total of seven proteins fractions have been detected from the haemolymph. The chemical nature of different protein fractions have been examined by histochemical methods. The changes taking place in the cuticle and epidermal cells have been examined during the transformation of 6th nymph into adult. The fat body proteins have been electrophoretically fractioned and the changes in the concentration of different protein fractions have been examined. It is suggested that the protein fraction 3 of the haemolymph is utilized in the formation of new cuticle. It is concluded by the histochemical observations that proteins of the band 3 are synthesized in the fat body.