Influence of Precocene II on the estimated changes within the haemocyte population of parasitized spodoptera littoralis larvae by microplitis rufiventris

Series of experiments were undertaken to investigate whether application of Precocene II (PII) to parasitized S. littoralis larvae can influence their THCs and DHCs. PII was applied in single dose treatments (70μg/5 μl/dose). Haemolymph samples were taken at daily intervals from day 3 to day 8 post‐treatments. Two distinct alterations were noted; first, PII‐treatments caused significant decrease in THCs. Once the parasitoid larvae emerge from either treated or untreated hosts, a sharp decline in THCs was observed. The second effect was changes in DHCs of S. littoralis larvae. In controls, haemocyte types could be arranged in descending order due to their relative occurrence into plasmatocytes (PLs), prohaemocytes (PRs), spherule cells (SPs), granular cells (GRs) and oenocytoids (OEs). This order changed in treated hosts to PLs, GRs, PRs, SPs and OEs, i.e., GRs that were less prevalent in the haemolymph of control larvae increased significantly in PII‐treated hosts. In the latters, sharp rise in GRs levels followed parasitoid emergence. OEs cells were scarce in both treated and untreated hosts. No significant difference was observed between the overall means of PLs in both types of host larvae. But GRs‐PRs was markedly altered in treated larvae.     

Fungal infection causes changes in the number,morphology and spreading ability of Galleria mellonella haemocytes

The most effective and important strategy in the insect immune response is based on cellular reactions incorporating haemocytes. The present study uses Galleria mellonella (Lepidoptera: Pyralidae) as a host to study the pathogenesis caused by the entomopthoralean fungus Conidiobolus coronatus (Entomophthorales). Five types of haemocytes with different morphologies and behaviour are observed in the haemolymph of G. mellonella: granulocytes (GRs), plasmatocytes (PLs), spherulocytes (SPs), oenocytes (OEs) and prohaemocytes (PRs). During in vitro cultivation, three morphological subtypes of PLs are distinguished: flattened PLs, sun‐like PLs and oval PLs. In fresh smears of haemolymph observed under phase‐contrast microscopy, only flattened PLs are identified. No morphological changes are observed between fresh smears and in vitro cultures for GR, OE, SP and PR. Haemocytes cultured in vitro form a cellular network composed of PLs and GRs. Changes in the numbers, morphology and behaviour of haemocytes induced by fungal infection are compared with those observed in normally‐developing untreated larvae. Infection results in a significant drop in the number of haemocyte types. Fresh smears of haemocytes from mycosed larvae reveal malformed OEs, vacuolized PLs and GRs, as well as PLs with apoptotic blebs. Haemocytes from mycosed larvae incubated in vitro look similar, with degranulated GRs and vacuolized PLs forming microaggregations, as well as deformed OEs; only the SPs remain unharmed. Fungal infection impairs the ability of haemocytes to attach and spread on the culture dish. The actin cytoskeleton of haemocytes from mycosed larvae appear disorganized.     

华北大黑鳃金龟幼虫血细胞的超微结构观察

应用光学和电子显微镜技术检查了华北大黑鳃金龟3龄幼虫血淋巴内的血细胞,识别出5种类型的血细胞(原血胞、浆血胞、颗粒血胞、珠血胞和凝血胞)并对每一种血细胞的超微结构特点进行描述。     

EFFECT OF PARASITIZATION BY THE PUPAL ENDOPARISITOID,PTEROMALUS PUPARUM (HYMENOPTERA: PTEROMALIDAE) ON HUMORAL IMMUNE REACTIONS OF PIERIS RAPAE (LEPIDOPETERA: PIERIDAE)*

Abstract Studies on the effect of parasitization by the endoparasitoid on host humoral immune reactions are carried out with the pupal endoparisitic wasp, Pteromalus puparum, and its host, Pieris rapae. Phenoloxidase (PO) activity of parasitized hosts hemolymph increased significantly at 12 h, day four and day five after parasitization. Hem‐agglutination activity of parasitized hosts hemolymph was always higher than that of wounded and unparasitized ones. Moreover, antibacterial activity of parasitized hosts hemolymph became more and more stronger, whilst wounded and unparasitized pupae only owned a weak antibacterial activity. It suggested that activities of humoral immune factors of Pieris rapae could be influenced to some degrees by P. puparum.     

菜粉蝶蛹血细胞体外细胞免疫反应及蝶蛹金小蜂毒液的影响

为探明蝶蛹金小蜂Pteromalus puparum毒液对其寄主菜粉蝶Pieris rapae蛹颗粒血细胞和浆血细胞的包囊与吞噬能力的影响, 本研究分别采用Na₂-EDTA处理和尼龙毛法对菜粉蝶蛹颗粒血细胞与浆血细胞进行分离纯化; 再采用离体细胞培养方法, 研究了菜粉蝶蛹颗粒血细胞、 浆血细胞各自在包囊和吞噬反应中的作用, 以及蝶蛹金小蜂毒液对其所产生的影响。结果表明： 颗粒血细胞和浆血细胞均参与了包囊反应, 其中前者包囊作用明显, 后者作用微弱, 但两者同时存在时包囊作用最为明显; 血淋巴浆质对颗粒血细胞和浆血细胞包囊反应均无显著影响。毒液对颗粒血细胞和浆血细胞的包囊能力均存在显著的抑制作用, 且抑制作用具有明显的剂量效应特征。此外, 菜粉蝶蛹颗粒血细胞和浆血细胞均具吞噬能力, 其中前者吞噬能力显著强于后者; 毒液对颗粒血细胞与浆血细胞的吞噬能力亦均存在显著的抑制作用, 且该抑制作用亦具有显著的剂量效应特征。结果说明, 菜粉蝶蛹颗粒血细胞及浆血细胞均参与寄主的细胞免疫反应, 蝶蛹金小蜂毒液对其寄主颗粒血细胞和浆血细胞的包囊与吞噬能力均存在显著的抑制作用。     

Response of the wasp (<Emphasis Type="Italic">Cotesia glomerata</Emphasis>) to larvae of the large white butterfly (<Emphasis Type="Italic">Pieris brassicae</Emphasis>)

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.     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

Isolation and characterization of an immunosuppressive protein from venom of the pupa-specific endoparasitoid Pteromalus puparum

In hymenopteran parasitoids devoid of symbiotic viruses, venom proteins appear to play a major role in host immune suppression and host regulation. Not much is known about the active components of venom proteins in these parasitoids, especially those that have the functions involved in the suppression of host cellular immunity. Here, we report the isolation and characterization of a venom protein Vn.11 with 24.1 kDa in size from Pteromalus puparum, a pupa-specific endoparasitoid of Pieris rapae. The Vn.11 venom protein is isolated with the combination of ammonium sulfate precipitation and anion exchange chromatography, and its purity is verified using SDS-PAGE analysis. Like crude venom, the Vn.11 venom protein significantly inhibits the spreading behavior and encapsulation ability of host hemocytes in vitro. It is suggested that this protein is an actual component of P. puparum crude venom as host cellular-immune suppressive factor.     

Venom of Pteromalus puparum (Hymenoptera: Pteromalidae) induced endocrine changes in the hemolymph of its host,Pieris rapae (Lepidoptera: Pieridae)

Pteromalus puparum is a predominant endoparasitoid wasp of Pieris rapae. Its venom is the only active factor injected into host associated with oviposition. In this report, we explored whether the venom alone from this wasp affects the endocrine system of its host or not. We monitored the changes of hemolymph juvenile hormone (JH; only JH III detected), ecdysteroid, and juvenile hormone esterase activity (JHE) over 72 h in parasitized and venom‐microinjected P. rapae pupae. Non‐parasitized and PBS‐microinjected P. rapae served as controls. Results showed that JH titers were significantly higher in parasitized and venom‐microinjected pupae than that in control pupae during 24 to 72 h. After 12 h, JH titers were significantly promoted by parasitization and venom microinjection. JHE activities of non‐parasitized and PBS‐microinjected pupae were significantly higher than that of parasitized and venom‐microinjected pupae, which was with a peak at 12 h (parasitized pupae) or 24 h (venom‐microinjected pupae) during 6 to 48 and 12 to 36 h, respectively. The hemolymph titers of ecdysteroid in non‐parasitized and PBS‐microinjected pupae increased rapidly during 12 to 36 h with a peak at 36 h, and were higher than treatments before 48 h, while presenting a significant difference at 24 to 48 h between the treatments and controls. The results demonstrate that venom alone of this parasitoid wasp can disrupt its host's endocrine system. © 2009 Wiley Periodicals, Inc.     

Pteromalus puparum venom impairs host cellular immune responses by decreasing expression of its scavenger receptor gene

Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Although there is a rich literature on these systems, parasitoid immune-disabling mechanisms have not been fully elucidated. Here we report on a newly discovered immune-disabling mechanism in the Pieris rapae/Pteromalus puparum host/parasitoid system. Because venom injections and parasitization suppresses host phagocytosis, we turned attention to the P. rapae scavenger receptor (Pr-SR), posing the hypothesis that P. puparum venom suppresses expression of the host Pr-SR gene. To test our hypothesis, we cloned a full-length cDNA of the Pr-SR. Multiple sequences alignment showed the deduced amino acid sequence of Pr-SR is similar to scavenger receptors of other lepidopterans. Bacterial and bead injections induced Pr-SR mRNA and protein expression, which peaked at 4 h post-bead injection. Venom injection inhibited Pr-SR expression. Pr-SR was specifically expressed in granulocytes compared to plasmatocytes. We localized the Pr-SR protein in cytoplasm and cellular membrane, with no evidence of secretion into host plasma. Double-strand RNA designed to Pr-SR mRNA silenced expression of Pr-SR and significantly impaired host phagocytosis and encapsulation reactions. Venom injections similarly silenced Pr-SR expression during the first 8 h post-treatment, after which the silencing effects gradually abated. We infer from these findings that one mechanism of impairing P. rapae hemocytic immune reactions is by silencing expression of Pr-SR.     

The ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) differentially affects cells mediating the immune response of its flesh fly host,Sarcophaga bullata Parker (Diptera: Sarcophagidae)

In this study, we examined cellular immune responses in the flesh fly, Sarcophaga bullata, when parasitized by the ectoparasitoid Nasonia vitripennis. In unparasitized, young pharate adults and third instar, wandering larvae of S. bullata, four main hemocyte types were identified by light microscopy: plasmatocytes, granular cells, oenocytoids, and pro-hemocytes. Parasitism of young pharate adults had a differential effect on host hemocytes; oenocytoids and pro-hemocytes appeared to be unaltered by parasitism, whereas adhesion and spreading behavior were completely inhibited in plasmatocytes and granular cells by 60 min after oviposition. The suppression of spreading behavior in granular cells lasted the duration of parasitism. Plasmatocytes were found to decline significantly during the first hour after parasitism and this drop was attributed to cell death. Melanization and clotting of host hemolymph did not occur in parasitized flies, or the onset of both events was retarded by several hours in comparison to unparasitized pharate adults. Hemocytes from envenomated flies were altered in nearly identical fashion to that observed for natural parasitism; the total number of circulating hemocytes declined sharply by 60 min post-envenomation, the number of plasmatocytes declined but not granular cells, and the ability of plasmatocytes and granular cells to spread when cultured in vitro was abolished within 1 h. As with parasitized hosts, the decrease in plasmatocytes was due to cell death, and inhibition of spreading lasted until the host died. Isolated crude venom also blocked adhesion and spreading of these hemocyte types in vitro. Thus, it appears that maternally derived venom disrupts host immune responses almost immediately following oviposition and the inhibition is permanent. The possibility that this ectoparasite disables host defenses to afford protection to feeding larvae and adult females is discussed.     

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT₇ genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT_1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.     

The reproductive strategy of the gregarious parasitoid,Pteromalus puparum (Hymenoptera: Pteromalidae)

Summary Pteromalus puparum is a gregarious parasitoid of lepidopterous pupae. To determine in which phase of a host outbreak superparasitism occurs, field investigations were carried out on seasonal prevalence of the host, Papilio xuhtus, and parasitism by P. puparum in a citrus grove in Fukuoka, Japan in 1974. Host pupation occurred from May to November and the parasitoid attacked the host throughout this period. Pupal density increased rapidly after mid-August and the percentage parasitism decreased during this period. A high level of parasitism was attained after one or more parasitoid generations in mid-September. The superparasitism was observed after mid-September when the parasitoid attained extremely high density, and resulted in an increase in the proportion of males, high mortality, and a decrease in the size of the progeny.     

Impact of flowering buckwheat on Lepidopteran cabbage pests and their parasitoids at two spatial scales

We assessed the potential of annual buckwheat, Fagopyrum esculentum Moench, to lead to improved parasitism of lepidopteran cabbage pests over four years. Pest, parasitism, and hyperparasitism rates were monitored in replicated cabbage plots (12 × 20 m) with or without 3 m wide buckwheat borders from 2000 to 2003. Floral borders did not significantly increase egg, larval, or pupal densities of cabbage looper, Trichoplusia ni (Hübner), imported cabbageworm, Pieris rapae (L.), or diamondback moth, Plutella xylostella (L.). Buckwheat increased parasitism rates by Voria ruralis (Fallen) on T. ni larvae and Cotesia rubecula (Marshall) on P. rapaelarvae over four years. Parasitism by Diadegma insulare (Cresson) on P. xylostella larvae was higher in buckwheat than control plots in the first year, and parasitism by Euplectrus plathypenae (Howard) on T. ni larvae was lower in buckwheat than control plots in the second year. The hyperparasitoid Conura side (Walker) attacked D. insulare all four years, but buckwheat did not affect hyperparasitism rates. The effect of spatial scale on pest densities and parasitism in 2001 was evaluated by comparing plots separated at least 67 m (nearby) versus 800 m apart (isolated). T. ni pupae and P. rapae eggs and pupae were more abundant in plots in closer proximity, whereas P. xylostella densities did not vary by the spatial separation of plots. Tachinids and Pteromalus puparum (L.) attacked more P. rapae in nearby plots. E. plathypenae responded to the treatment × scale interaction, parasitizing more in control than buckwheat when plots were isolated but not when plots were nearby.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by a pupal endoparasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae): effects of parasitization and venom on host hemocytes

In contrast to the situation with egg-larval and larval endoparasitic wasps, little is known about the effects of pupal endoparasitoids and their secretions on the hemocytes of their insect hosts. This study focuses on the pupal endoparasitoid, Pteromalus puparum, and its host, the small white butterfly, Pieris rapae. Parasitism by P. puparum, resulted in a significant increase in the total number of host hemocytes up to day five after parasitization. From day one to day four after parasitization, the percentage of plasmatocytes significantly decreased, and the proportion of granular cells increased. Moreover, from 12 h to day three after parasitization, hemocyte mortality in parasitized pupae was noticeably higher. When P. rapae pupae were parasitized by adult females of P. puparum irradiated by gamma-ray (pseudoparasitization), it was clear that the treated wasps could induce similar hemocyte changes. However, such phenomena did not occur in punctured host pupae (mimic-parasitization). After treatment with P. puparum venom, both the percentages of spreading plasmatocytes and encapsulated Sephadex G-25 beads were lessened significantly in vitro. Electron microscopy analysis and visualization of hemocyte F-actin with phalloidin-FITC showed that hemocytes treated with venom had a rounded configuration and neither spread nor extended pseudopods, while there was no marked alteration of hemocyte cytoskeletons after venom treatment. The results suggested that venom of P. puparum could actively suppress the hemocyte immune response of its host, but not by destroying the host hemocyte cytoskeleton.     

Aphelinus albipodus(Hymenoptera: Aphelinidae) andDiaeretiella rapae(Hymenoptera: Braconidae) Parasitism onDiuraphis noxia(Homoptera: Aphididae) Infesting Barley Plants Differing in Plant Resistance to Aphids

Russian wheat aphid,Diuraphis noxia(Mordvilko), as a pest of small grains, has prompted research into biological control and host plant resistance. In the presence of Russian wheat aphid, leaves of a susceptible barley (Morex) are curled and chlorotic and sustain large densities of this aphid, while leaves of a resistant barley (STARS-9301B) remain flat and green and sustain fewer aphids. Might parasitism of Russian wheat aphid byAphelinus albipodusHayat & Fatima andDiaeretiella rapaeMcIntosh be affected differently by these plant types? When presented the plants separately and based on parasitism rate relative to aphid density, the largerD. rapaewas more effective in parasitizing relatively high densities of aphids within curled leaves of Morex than relatively low densities of aphids on uncurled leaves of STARS-9301B. Parasitism byA. albipodusdid not significantly differ among the plants. When given a choice of plants, approximately equal rates of parasitism occurred on the two plant lines for both parasitoid species, and parasitism byD. rapaewas greater thanA. albipodus.These data indicate that using parasitoid size as an indicator of success in a physically restricted environment may be misleading, when considered in a plant environment responsive in several manners to aphids (chlorosis, curling, and ability to sustain Russian wheat aphid). We expect that use of resistant barley will result in decreased parasitoid abundance as aphid densities decrease. However, parasitism rates are expected to be approximately equal on resistant and susceptible barley. In this system, plant resistance and biocontrol are compatible management strategies.     

Dynamic effects of parasitism by an endoparasitoid wasp on the development of two host species: implications for host quality and parasitoid fitness

1. The study reported here examined growth and developmental interactions between the gregarious larval koinobiont endoparasitoid Cotesia glomerata (Hymenoptera: Braconidae) and two of its hosts that vary considerably in growth potential: Pieris rapae and the larger P. brassicae (Lepidoptera: Pieridae). At pupation, healthy larvae of P. brassicae are over twice as large, in terms of fresh body mass, as those of P. rapae. 2. Clutch size of C. glomerata was manipulated artificially, and the relationship between parasitoid burden and the maximum weight of the parasitised host (= host–parasitoid complex) was measured. In both hosts, the maximum complex weight was correlated positively with parasitoid burden. Compared with unparasitised hosts, however, the growth of P. rapae was increased at significantly lower parasitoid burdens than in P. brassicae. Emerging wasp size was correlated negatively with parasitoid burden in both host species, whereas development time was less affected. 3. After larval parasitoid egress, the weight of the host carcass increased slightly, but not significantly, with parasitoid burden, although there was a strong correlation between the proportion of host mass consumed by C. glomerata larvae during development and parasitoid burden. 4. Clutch size was generally correlated positively with instar parasitised in both hosts, and greater in P. brassicae than in P. rapae. Sex ratios were much more female biased in L1 and L2 P. rapae than in all other host classes. Adult parasitoid size was correlated inversely with host instar at parasitism, and wasps emerging from P. brassicae were larger, and completed development faster, than conspecifics emerging from P. rapae. 5. The data reveal that parasitism by C. glomerata has profound species‐specific effects on the growth of both host species. Consequently, optimality models in which host quality is often based on host size at parasitism or unparasitised growth potential may have little utility in describing the development of gregarious koinobiont endoparasitoids. The results of this investigation are discussed in relation to the potential effectiveness of gregarious koinobionts in biological control programmes.