Host regulation and release of parasitism-specific proteins in the system Toxoneuron nigriceps-Heliothis virescens

The braconid wasp Toxoneuron nigriceps induced qualitative and quantitative changes in the protein composition of the moth Heliothis virescens host hemolymph. Total protein concentration was found to be higher in parasitized host 4 days after parasitism as compared to control hosts, mainly due to changes in a particular group of proteins. Host proteins with a molecular mass of 173 and 72 kDa were found in higher levels in the hemolymph of parasitized larvae as control hosts approached pupation, while an 80 kDa peptide was found in reduced concentration in the hemolymph of parasitized hosts. Levels of these three peptides were maintained throughout parasitoid development, while two of them (173 and 72 kDa) were cleared from the host hemolymph close to pupation. Besides the regulation of host proteins, three parasitism-specific proteins (PSPs) were released into the host hemolymph. Two of them (PSP1-MW=116 kDa, pI=6.3; PSP2-MW=114 kDa, pI=6.2) first appeared in the hemolymph of parasitized hosts soon after pupation of control host and increased in concentration as the parasitoid developed. The third PSP (PSP3-MW=56 kDa, pI=5.8) was produced towards the end of parasitoid larval development, close to parasitoid egression. Database searches based on the amino acid composition and amino terminal sequence of PSP1 and PSP2 did not produce any significant matches, while PSP3 was identified as a putative chitinase. Incubation of host derived tissues, parasitoid larvae and teratocytes in 35S conditioned media suggested PSPs were a product of teratocytes. The role of the regulation of host proteins and release of PSPs by teratocytes for the successful development of T. nigriceps are discussed.     

Teratocytes growth pattern reflects host suitability in a host–parasitoid assemblage

Abstract.  In some parasitoid species, the serosa membrane breaks apart at hatching and produces teratocyte cells that assume various functions (immunossupression, secretion and nutrition) mediating host–parasitoid relationships. Teratocyte growth pattern may thus reflect the host suitability for a parasitoid. The teratocyte growth pattern (increase in size and number of teratocytes as a function of time) is studied and used as an indirect measure of fitness to compare the development of the endoparasitoid Dinocampus coccinellae in a marginal host, the coccinellid Harmonia axyridis , and in a suitable host, Coleomegilla maculata . Indirect measures of fitness recorded in both host species confirm that C. maculata is a suitable host for D. coccinellae contrary to the marginal host H. axyridis. According to regression analysis, teratocyte numbers decrease linearly whereas teratocyte size increases linearly with time in the suitable host C. maculata (larvae or adults). In the marginal host, parasitism occurs only in the larval stage where a delay in the parasitoid larval development is observed. Increase in teratocyte size is also highly variable. The teratocyte growth pattern of the parasitoid in the marginal host does not follow the linear model found in the suitable host. Teratocyte growth pattern may be a useful criterion to evaluate host-suitability and host range of parasitoids.     

Dinocampus (=Perilitus) coccinellae teratocyte-specific polypeptide: its accumulative property,localization and characterization

Dinocampus (=Perilitus) coccinellae (Braconidae: Hymenoptera) teratocytes synthesize a teratocyte-specific polypeptide (TSP) with a high molecular weight of 540kDa. The TSP has a tendency to accumulate in the teratocyte cells without release after synthesis ([Okuda and Kadono-Okuda, 1995]), which was confirmed in this study. Pulse-chase fluorography indicated that teratocytes at a younger stage (6 days after parasitization)secreted negligible TSP into the medium after synthesis, while teratocytes at an older stage (11 days after parasitization)secreted the synthesized products into the medium, although the amount released was still low. Western blot with anti-TSP serum showed that only a small amount of TSP appeared in the parasitized host hemolymph, even when TSP synthesis by teratocytes was actively taking place, which also supported the accumulative nature of TSP. The immunoelectronmicroscopic studies revealed that the TSP was localized specifically in high electron-dense vacuoles. Lectin blot analysis identified TSP as a high mannose glycoprotein. The amino acid composition of the major subunit of the TSP was quite similar to that of nutritive proteins such as vitellogenin and storage proteins of some insects. These characterization data, together with the accumulation property of the TSP indicates that Dinocampus teratocyte primarily plays a nutritive role for the developing parasitoid larvae. TSP exhibited esterase activity, which indicates that TSP may have an additional function in the host-parasitoid reaction.     

Host regulation by the aphid parasitoid Aphidius ervi: the role of teratocytes

The biochemical profile and metabolism of Acyrthosiphon pisum(Harris) (Homoptera, Aphididae) are markedly altered and redirected in response to parasitization by the endophagous braconid Aphidius erviHaliday (Hymenoptera, Braconidae). In the present study, the role played in the host regulation process by teratocytes, cells deriving from the dissociation of the embryonic membrane of the parasitoid, is taken into consideration. The protein synthesis activity of these cells of embryonic origin is analysed in vitroand an essential characterization of those proteins de novosynthesized and released in the incubation medium is provided. Teratocytes, obtained by dissecting parasitized host aphids, 3, 4 and 5 days after parasitoid oviposition, were incubated in vitroand, at the end of the incubation period, were separated from the medium for SDS-PAGE analysis of both cellular and secreted proteins. Various cellular proteins were more abundant as the time between parasitization and teratocyte collection increased. Furthermore, two proteins, showing an approximate molecular mass of 15 kD (p15) and 45 kD (p45) respectively, were abundantly secreted in the incubation medium by 5 day-old teratocytes. Incubations in presence of ³⁵S radiolabelled amino acids indicated that p15 and p45 are both synthesized by A. erviteratocytes. The amino acid composition of these two proteins was similar to that reported for other insect proteins with a demonstrated nutritional function. The p45 protein was found to be glycosylated. A tentative physiological model describing the host regulation role played by different parasitoid-derived factors is proposed.     

Functional analysis of a fatty acid binding protein produced by Aphidius ervi teratocytes

Aphidius ervi (Hymenoptera, Braconidae) is an endophagous parasitoid of various aphid species, including Acyrthosiphon pisum (Homoptera, Aphididae), the model host used in the present study. Parasitized hosts show a marked increase of their nutritional suitability for the developing parasitoid larvae. This alteration of the biochemical and metabolic profile is due to a castration process mediated by the combined action of the venom, injected at the oviposition, and of the teratocytes, cells deriving from the dissociation of the embryonic membrane. Teratocytes produce and release in the host haemocoel two parasitism-specific proteins, which are of crucial importance for the development of their sister larvae. One of the proteins is a fatty acid binding protein (Ae-FABP), which shows a high affinity for C14-C18 saturated fatty acids (FAs) and for oleic and arachidonic acids. To better define the possible nutritional role of this protein, we have studied its immunolocalization profile in vivo and the impact on FA uptake by the epidermal and midgut epithelia of A. ervi larvae. During the exponential growth of A. ervi larvae, Ae-FABP is distributed around discrete lipid particles, which are abundantly present in the haemocoel of parasitized host aphids and in the midgut lumen of parasitoid larvae. Moreover, a strong immunodetection signal is evident on the surface of the two larval epithelia involved in nutrient absorption: the parasitoid midgut epithelium and the external epidermal layer. These two epithelia can effectively absorb radiolabelled myristic acid, but the FA transport rates are not affected by the presence in the medium of Ae-FABP. The protein appears to act essentially as a vector in the host haemolymph, transferring FAs from the digestion sites of host lipids to the growing parasitoid larvae. These data indicate that the proteins produced by A. ervi teratocytes may play complementary roles in the nutritional exploitation of the host.     

畸形细胞在协调寄生蜂同其寄主 相互关系中的作用

膜翅目内寄生的茧蜂科(Ｂｒａｃｏｎｉｄａｅ)、广腹细蜂科(Ｐｌａｔｙｇａｓｔｅｒｉｄａｅ)、缘腹卵蜂科(Ｓｃｅｌｉｏｎｉｄａｅ)中,蜂卵孵化时,起胚胎营养作用的浆膜(ｓｅｒｏｓａｌｍｅｍｂｒａｎｅ)释放到寄主血淋巴中,分散后游离形成畸形细胞。赤眼蜂科(Ｔｒｉｃｈ...     

畸形细胞在协调寄生蜂同其寄主 相互关系中的作用

Teratocytes originating from the serosal membrane of an endoparasitic wasp embryo, which is one of the species found in the Hymenopteran families Braconidae, Platygasteridae and Socelionidae, act an important role in coordinating the relationship between the larval wasp and its host. It may be concluded that inhibiting the immune system of the host, regulating development of the host and nourishing the parasitoid larva are three major functions of the teratocytes. By means of releasing inhibitor of phenol oxidase, a fundamental ingredient of the immune system of the host, the teratocytes prevent the host from encapsulation, which is fatal to the parasitoid larva. In the process of parasitism, the teratocytes reduce ecdysteroid titer and inhibit the metabolism of JH in the hemoymph of the host, and subsequently result in the developmental arrest of the host larva. On the other hand, the teratocytes secrete proteins or present themselves as food to the parasitoid. Although all those above are generally acknowledged functions of the teratocytes, there are other putative functions of the teratocytes, such as secreting histolytic enzymes and mycostatic material, and protecting the host from overharmed by the parasitoid before the parasitoid emerges from its host. The studies in this area can provide us more understanding and knowledge about the relationship between a parasitoid and its host.     

Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family

A cDNA clone was isolated from a rat liver lambda gt11 expression library by screening with polyclonal antibodies raised against a rat liver microsomal carboxylesterase. This clone of 1.8 kb contained an open reading frame encoding a mature protein of 531 amino acids with a predicted molecular weight of 58,084. The 5' portion of the clone coded for 9 amino acids of a putative signal peptide. The 3' end of the clone included an untranslated region and a poly (A) tail. Carboxylesterase active site regions, five potential N-linked glycosylation sites, and 2 postulated cystine disulfide bridges were found in the cDNA-deduced amino acid sequence. Sequences obtained from tryptic peptides and the NH2-terminus of the purified native carboxylesterase were aligned with the deduced amino acid sequence, and the overall identity was 84%. Southern blot analysis suggested the presence of multiple genes. Thus it is concluded that we have cloned a rat liver carboxylesterase, and that this enzyme is a member of a multigene family.     

寄主龄期、过寄生和寄主饥饿处理对菜蛾盘绒茧蜂幼蜂及畸形细胞发育的影响

过寄生、寄生时寄主龄期和寄生后寄主饥饿处理影响菜蛾盘绒茧蜂Cotesia plutellae（Kurdj.）幼蜂及畸形细胞的发育。显微解剖和观察表明,4龄小菜蛾Plutella xylostella L.幼虫被寄生后,其体内菜蛾盘绒茧蜂幼蜂发育不整齐、假寄生比例增高。过寄生后,每头被寄生的寄主血腔中畸形细胞数量明显增多,但直径变小;随着过寄生程度的加剧,幼蜂发育严重受阻。寄主营养显著影响体内幼蜂及畸形细胞的发育,被寄生的小菜蛾经饥饿处理62 h后,体内畸形细胞的数量、活性明显降低,与此同时,幼蜂的发育也受到明显抑制,寄主发育与寄生蜂和畸形细胞的发育呈正相关性。由此可见,寄主不同龄期、过寄生及寄主营养状况均对寄主体内幼蜂和畸形细胞发育产生影响。     

Larvae of an endoparasitoid,Cotesia kariyai (Hymenoptera: Braconidae), feed on the host fat body directly in the second stadium with the help of teratocytes

Larvae of a gregarious endoparasitoid, Cotesia kariyai (Watanabe), grew rapidly during the second stadium in the host. The fat body of a Pseudaletia host parasitized by C. kariyai was completely consumed by 10 d, just before larval emergence. It seemed hard to explain the growth of the second instar parasitoids and the rapid consumption of the fat body only by ingestion of hemolymph converted from the fat body or other organs of the host. Paraffin sections of the parasitized host revealed that many teratocytes were attached to the surface of the fat body in many sites and destroyed the fat body tissue locally. Zymography of proteins released from the teratocytes revealed that the teratocytes 4 to 9 days after parasitization showed collagenase activity (as a gelatinase). Further, 1st instar parasitoids which were transplanted together with teratocytes into unparasitized hosts preconditioned with C. kariyai polydnavirus (CkPDV) plus venom, grew normally to the 2nd stadium. Abnormal growth of parasitoid larvae was observed when parasitoid larvae were transplanted without teratocytes. These results suggest that the teratocytes attach to the outer sheath of the fat body, secrete an enzyme that makes a hole in the matrix of the fat body, thus allowing the second instar parasitoid to ingest the content of the fat body.     

一种中红侧沟茧蜂畸形细胞分泌的寄生特异蛋白

SDS-PAGE电泳表明,黏虫Pseudaletia separa-ta、棉铃虫 Helicoverpa armigera、小地老虎 Agrotisypsilon的幼虫受中红侧沟茧蜂Microplitis mediator寄生后,血淋巴中都出现一个98.6 kDa的寄生特异蛋白(p98.6)。畸形细胞(teratocytes)的体外培养发现,p98.6是由来自中红侧沟茧蜂胚胎浆膜层的畸形细胞分泌的。这一结果将为研究寄生蜂的寄生生理和畸形细胞在协调寄生蜂和寄主关系中的作用打下基础。     

Growth patterns of Microplitis rufiventris (Hym., Braconidae) teratocytes in Spodoptera littoralis (Lep., Noctuidae) larvae treated with a chitin synthesis inhibitor

The influence of sublethal dietary levels (0.4 p.p.m.) of a chitin synthesis inhibitor lefenuron { N -[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenylaminocarbonyl]-2,6-difluorobenzamide} on the development of Microplitis rufiventris teratocytes was investigated. Observations and data were taken on maturing teratocytes, i.e. at the end of parasitoid development. When embryogenesis of the parasitoid eggs was initiated in treated hosts, the dissociated cells of the embryonic membrane of some of the parasitoid larvae did not liberate in the haemolymph of the host, whereas in the other hosts, the released ones as individual cells scored the largest mean number per host larva. When young teratocytes were exposed to the treatment they were found in some cases clumping in masses or clustered around the parasitoid larvae. In treated hosts, different size classes of teratocytes co-existed. Larger and morphological abnormal cells were more common in hosts which contained older teratocytes at the time of treatments. Abnormal teratocytes were associated with developmental abnormalities of parasitoid larvae, possibly due to deterioration of the host environment. This observation was common in treated hosts and in few cases of untreated ones. A significant decrease in teratocyte size occurred 24 h after parasitoid emergence. Observations and results obtained in the present work suggested that: (1) teratocyte may function in controlling the growth and successful development of parasitoid larvae; (2) they might have a protective function through sequestering abnormal materials issuing in the haemolymph of the host either during the course of parasitoid development or due to external stress; and (3) number of teratocyte cells liberated from the embryonic membrane of parasitoid egg is not constant.     

菜蛾盘绒茧蜂对寄主小菜蛾幼虫营养的调节和利用

寄主小菜蛾Plutella xylostella被内寄生蜂菜蛾盘绒茧蜂Cotesia plutellae寄生后,其取食、发育及营养代谢在各种寄生因子的作用下伴随幼蜂的发育而发生很大的变化,畸形细胞作为调节因子之一也发挥了重要的作用。本实验通过比较被寄生和未被寄生小菜蛾血淋巴蛋白浓度以及两种血淋巴对菜蛾盘绒茧蜂幼蜂进行体外培养的培养液的蛋白浓度,发现被寄生小菜蛾血淋巴比未被寄生小菜蛾血淋巴的蛋白浓度略低但差异不显著,而未被寄生小菜蛾血淋巴幼蜂培养液的蛋白浓度显著低于被寄生小菜蛾血淋巴幼蜂培养液的蛋白浓度,证明畸形细胞的蛋白质分泌功能。被寄生后期, 小菜蛾体重明显大于未被寄生的小菜蛾体重,而脂肪体重量相比正好相反;通过显微染色观察,在小菜蛾念珠状脂肪体表面粘附有畸形细胞,对脂肪体进行分解破坏而使其成颗粒状; 蛋白含量和脂滴浓度测定也表明,脂肪体的可溶性蛋白含量和脂滴浓度也迅速降低,同比低于未被寄生小菜蛾。而与此同时,幼蜂正处在快速生长阶段,中肠酯酶的活性逐步上升,幼蜂得以快速消化吸收小菜蛾体内的营养直到完成幼虫发育,整个幼蜂的脂滴浓度也达到了最大值。因此寄生后期,推测在畸形细胞的协助下,幼蜂吸收了寄主小菜蛾体内的营养为自身生长发育所用。     

Teratocytes: Growth and numbers in the hemocoel of Heliothis virescens attacked by Microplitis croceipes

When the egg of Microplitis croceipes hatches in its host, Heliothis virescens, spherical cells (teratocytes) from the extraembryonic membrane are released into the host's hemolymph. Approximately 750 teratocytes are liberated from the parasitoid egg, and they average 10.5 μm in diameter when released. These cells increase in size, reaching a maximum average diameter of 140 μm in 8–9 days.The developing parasitoid emerges from the host in 9 days. The host remains alive and contains approximately 750 teratocytes, indicating that the teratocytes are not consumed by the parasitoid. These results suggest that teratocytes are not a direct source of nutrition for the developing parasitoid.Observations showed that encapsulation can occur in the presence of teratocytes even following the emergence of the parasitoid. The results indicate that teratocytes do not block the host's ability to encapsulate certain foreign materials within its hemocoel.     

Possible bases of pseudoparasitism in Spodoptera littoralis larvae stung by Microplitis rufiventris

The effects of host age and parasitoid female age on the occurrence of 'Pseudoparasitism', using the Spodoptera littoralis-Microplitis rufiventris host-parasitoid system were investigated. The first four larval instars of the host are not equally suitable for parasitoid development. The proportion of pseudoparasitized hosts significantly increases when: (1) the age of the female parasitoid increases; (2) oviposition occurs mostly in fourth instar larvae; (3) a later age of the host instar is used; (4) the mandibles of the newly hatched parasitoid larvae mistakenly attack host interior organs (e.g. Malpighian tubules); and (5) an imperfect growth pattern of teratocytes occurs. The reluctance of female wasps to parasitize fourth instar host larvae is not due to the thickness of host cuticle but possibly due to the unfavourable physiological state of the host larvae. The age of host larvae at the time of parasitization may influence the adverse effects of parasitoid factors (e.g. polydnavirus, venom and teratocytes) on the growth of host larvae. It is suggested that females of M. rufiventris are able to determine the suitability of a potential host instar for the development of their offspring. The cell diameter of M. rufiventris teratocytes increases with increasing age of host larvae at the time of oviposition. The association within the host of living parasitoid larvae and functional teratocytes may be important for the survival of each other and consequently for successful parasitism.     

Quantification and development of teratocytes in novel-association host-parasitoid combinations

We studied the development of teratocytes derived from two congeneric gregarious endoparasitic species, Cotesia chilonis and C. flavipes, parasitizing two congeneric novel hosts, Diatraea saccharalis and D. grandiosella. The host-parasitoid combinations studied allowed us to investigate relationships between host suitability and teratocyte development. D. saccharalis was a suitable host for both parasitoids, whereas D. grandiosella was suitable for C. chilonis development but often encapsulated C. flavipes progeny. Encapsulation of C. flavipes by D. grandiosella commenced around the time of parasitoid egg hatch, when teratocytes were released into the host's hemolymph. The gregarious parasitoids studied here released about 200 teratocytes per egg. Both absolute and normalized (teratocytes/parasitoid) numbers decreased over time. D. saccharalis supported more C. flavipes-derived teratocytes than D. grandiosella, possibly because of the unsuitability of the latter host. On intermediate assay days the number of C. flavipes-derived teratocytes was greater than for C. chilonis. However, C. chilonis-derived teratocytes grew larger than C. flavipes. Teratocytes in all host-parasitoid combinations doubled in size during parasitoid development. Teratocytes generally grew larger in D. grandiosella, which was a less suitable host.     

Effect of mating status and age of Microplitis rufiventris (Hym., Braconidae) females on the growth pattern and number of their teratocytes

The effect of mating status and age of the female of Microplitis rufiventris parasitoid on the growth pattern of its teratocytes during the larval development is described as well as the changes in the number of these cells. The growth pattern of teratocytes derived from haploid eggs significantly differed from that of eggs deposited by mated females. Both cell diameter and the number of maturing M. rufiventris teratocytes was fertilization-dependent. Following the parasitization of Spodoptera littoralis larvae with virgin parasitoid females, the host larvae hosted a large number of cells of smaller size at the end of parasitoid development. The opposite effect was seen when the host larvae contained female parasitoid larvae. The age of the female at parasitization had an effect on the maximum cell size attained prior to parasitoid emergence. It seems that there are two factors other than mating which affect the number of teratocytes: degeneration inside the host haemolymph and to some degree accidental ingestion by parasitoid larva.     

Low parasitoid success on a myrmecophilous host is maintained in the absence of ants

Abstract.  1. Studies of Dinocampus coccinellae , a parasitoid of ladybird beetles, have generally shown congruence between field parasitism rates of different host species and parasitoid preference and/or host suitability in the laboratory, suggesting that host intrinsic factors rather than habitat-related extrinsic factors are of greatest importance in determining D. coccinellae occurrence.
2. The myrmecophilous Coccinella magnifica exhibits much lower D. coccinellae prevalence in the field than most other Coccinella species: it has been suggested that this is a manifestation of enemy-free space provided by the predatory Formica rufa group ants with which the C. magnifica occurs.
3. Coccinella magnifica collected at the same time and locality as parasitised Coccinella septempunctata were unparasitised by D. coccinellae . In the laboratory, in the absence of ants, although the parasitoid attacked C. magnifica as readily as C. septempunctata , C. magnifica was not parasitised successfully.
4. Such results are consistent with those from other ladybirds and C. magnifica does not now benefit directly from any putative D. coccinellae -free space provided by aggressive ants. Because its close relatives exhibit high levels of D. coccinellae parasitism, C. magnifica may be useful in determining some elements important in the evolution of host protection against parasitoid attack.     

Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae).

Carboxylesterase-like enzyme cDNAs have been cloned and sequenced from malathion-resistant and susceptible strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). The cDNAs consist of 1963 nucleotides including a 35 bp untranslated 5'-end, a 1596 bp open reading frame, and a 332 bp untranslated 3'-end. The open reading frame encodes 532 amino acid residues. The predicted protein sequence from these cDNAs includes 2 potential N-glycosylation sites, a carboxylesterase type-B serine active site FGGDSENVTIFGESAG, and conserved residues Ser187, Glu317, and His432 to function as the catalytic triad. The predicted carboxylesterase-like enzyme sequence is most similar to that of the carboxylesterase from the peach-potato aphid, Myzus persicae with 45% sequence identity. Alignment of the parasitoid carboxylesterase-like enzyme cDNAs revealed that there are two nucleotide differences in the open reading frame between the parasitoid strains, including a silent mutation and a point mutation that presumably causes a gene product difference. A nucleotide thymine at position 658 in the susceptible strain cDNA is replaced by a guanine in the resistant strain cDNA. This substitution leads to an amino acid change from tryptophan (Trp220) in the susceptible strain to glycine (Gly220) in the resistant strain. This substitution is genetically linked to resistance but it is not known how or if this amino acid substitution affects detoxification of malathion. Northern blot analyses demonstrated that expression level of the carboxylesterase-like enzyme mRNA in adult A. calandrae is approximately 30-fold higher in the resistant strain relative to that in the susceptible strain. Southern analysis indicated that Pst I or Eco RI restriction sites are different in the two strains. Both a modified gene structure and an increase in expression of carboxylesterase may be responsible for the high level of resistance found in this beneficial wasp.