Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym : Braconidae)

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparositoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidose. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C rubecula PDV-protein (Crp32). Arch. Insect Biochem. Physiol. 53:92-100, 2003. (C) 2003 Wiley-Liss, Inc.

Virus or not? Phylogenetics of polydnaviruses and their wasp carriers

Our current, still limited, understanding of the comparative biology and evolution of polydnaviruses (PDVs) is reviewed, especially in the context of the possible origins of these parasitoid viruses and of their coevolution with carrier wasps. A hypothetical scenario of evolution of PDVs from ascovirus (or ascovirus-like) ancestors is presented, with examples of apparent extant transitional forms. PDVs appear, in the case of bracoviruses, to show phylogenetic relationships that mirror those of their wasp carriers: with ichno-viruses, the picture is less clear. Ongoing sequencing studies of entire PDV genomes from diverse wasp species are likely to greatly contribute to our understanding of PDV evolution. (C) 2003 Elsevier Science Ltd. All rights reserved.

Persistence and expression of Cotesia congregata polydnavirus in host larvae of the tobacco hornworm, Manduca sexta

The gregarious braconid wasp Cotesia congregata parasitizes host larvae of Manduca sexta, and several other sphingid species. Parasitism induces host immunosuppression due to the disruptive action of the wasp's polydnavirus (PDV) on host blood cells. During the initial stages of parasitism, these cells undergo apoptosis followed by cell clumping, which clears the hemolymph of a large number of cells. In this study, the persistence and expression of Cotesia congregata PDV (CcPDV) were examined using Southern and Nor-them blots, respectively. Digoxygenin-labelled total polydnaviral DNA was used to probe genomic DNA isolated from fat body and brains of hosts with emerged wasps taken 6 days following egress of the parasitoids, and significant cross-hybridization between the host fat body genomic DNA with viral DNA was seen. Thus, the virus persists in the host for the duration of parasitism. even during the post-emergence period, and may even be integrated in the host caterpillar DNA. Viral gene expression was examined using Northern blots and probes to the Cotesia rubecula CrV1 homolog, and the CrV1-like mRNAs were expressed as early as 4 h post-parasitization for at least 72 h and faint hybrization is even seen at the time the wasps eclose. In contrast, in Pieris rapae larvae the CrV1 transcript is expressed only for a brief time, during which time hemocyte function is disrupted. The effect is transitory, and hemocytes regain their normal functions after the parasites emerge as first instars. The genome of CcPDV contains one copy of the CrV1-like homolog as shown on Southern blots of viral genomic DNA. In conjunction with our earlier studies of the PDV-encoded early protein 1, the current work suggests multiple viral transcripts are produced following parasitization of the host. and likely target host hemocytes to induce their apoptosis, thereby preventing encapsulation of the parasitoid's eggs. Whether viral DNAs are integrated in the host's genomic DNA remains to be proven, but our results provide preliminary evidence that viral DNAs are detected in the host's fat body cells examined at the time of wasp ernergence and several days later. (C) 2003 Elsevier Science Ltd. All rights reserved.

Variation in the dispersal potential of non-feeding invertebrate larvae: the desperate larva hypothesis and larval size

For many species of marine invertebrates, variability in larval settlement behaviour appears to be the rule rather than the exception. This variability has the potential to affect larval dispersal, because settlement behaviour will influence the length of time larvae are in the plankton. Despite the ubiquity and importance of this variability, relatively few sources of variation in larval settlement behaviour have been identified. One important factor that can affect larval settlement behaviour is the nutritional state of larvae. Non-feeding larvae often become less discriminating in their 'choice' of settlement substrate, i.e. more desperate to settle, when energetic reserves run low. We tested whether variation in larval size (and presumably in nutritional reserves) also affects the settlement behaviour of 3 species of colonial marine invertebrate larvae, the bryozoans Bugula neritina and Watersipora subtorquata and the ascidian Diplosoma listerianum. For all 3 species, larger larvae delayed settlement for longer in the absence of settlement cues, and settlement of Bugula neritina larvae was accelerated by the presence of settlement cues, independently of larval size. In the field, larger W subtorquata larvae also took longer to settle than smaller larvae and were more discriminating towards settlement surfaces. These differences in settlement time are likely to result in differences in the distance that larvae disperse in the field. We suggest that species that produce non-feeding larvae can affect the dispersal potential of their offspring by manipulating larval size and thus larval desperation.

Humans, dogs and parasitic zoonoses - Unravelling the relationships in a remote endemic community in Northeast India using molecular tools

Canine parasitic zoonoses pose a continuing public health problem, especially in developing countries and communities that are socioeconomically disadvantaged. Our study combined the use of conventional and molecular epidemic, logical tools to determine the role of dogs in transmission of gastrointestinal (GI) parasites such as hookworms, Giardia and Ascaris in a parasite endemic teagrowing community in northeast India. A highly sensitive and specific molecular tool was developed to detect and differentiate the zoonotic species of canine hookworm eggs directly from faeces. This allowed epidemiological screening of canine hookworm species in this community to be conducted with ease and accuracy. The zoonotic potential of canine Giardia was also investigated by characterising Giardia duodenalis recovered from humans and dogs living in the same locality and households at three different loci. Phylogenetic and epidemiological analysis provided compelling evidence to support the zoonotic transmission of canine Giardia. Molecular tools were also used to identify the species of Ascaris egg present in over 30% of dog faecal samples. The results demonstrated the role of dogs as a significant disseminator and environmental contaminator of Ascaris lumbricoides in communities where promiscuous defecation practices exist. Our study demonstrated the usefulness of combining conventional and molecular parasitological and epidemiological tools to help solve unresolved relationships with regards to parasitic zoonoses.

A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph

Activation of prophenoloxidase (proPO) in insects is a defense mechanism against intruding microorganisms and parasites. Pattern recognition molecules induce activation of an enzymatic cascade involving serine proteinases, which leads to the conversion of proPO to active phenoloxidase (PO). Phenolic compounds produced by pPO-activation are toxic to invaders. Here, we describe the isolation of a venom protein from the parasitoid, Cotesia rubecula, injected into the host, Pieris rapae, which is homologous to serine proteinase homologs (SPH). The data presented here indicate that the protein interferes with the proteolytic cascade, which under normal circumstances leads to the activation of proPO and melanin formation. (C) 2003 Elsevier Ltd. All rights reserved.

Effects of settler size and density on early post-settlement survival of Ciona intestinalis in the field

Effects of variation in larval quality on post-metamorphic performance in marine invertebrates are increasingly apparent. Recently, it has been shown that variation in offspring size can also strongly affect post-settlement survival, but variation in environmental conditions can mediate this effect. The quality of habitat into which marine invertebrate larvae settle can vary markedly, and 1 influence on quality is the number of conspecifics present. We tested the effects of settler size and settler density on early (1 wk after settlement) post-settlement survival in the field for the solitary ascidian Ciona intestinalis. Larger settlers survived better than smaller settlers, within and among groups of siblings. Increases in the density of settlers decreased survival, but the density-dependent effects were much stronger for smaller settlers. We suggest that larger settlers are better able to cope with intra-specific competition because they have greater energetic reserves or a greater capacity to feed than smaller settlers.

Offspring size affects the post-metamorphic performance of a colonial marine invertebrate

The positive relationship between offspring size and offspring fitness is a fundamental assumption of life-history theory, but it has received relatively little attention in the marine environment. This is surprising given that substantial intraspecific variation in offspring size is common in marine organisms and there are clear links between larval experience and adult performance. The metamorphosis of most marine invertebrates does not represent a newbeginning, and larval experiences can have effects that carry over to juvenile survival and growth. We show that larval size can have equally important carryover effects in a colonial marine invertebrate. In the bryozoan Bugula neritina, the size of the non-feeding larvae has a prolonged effect on colony performance after metamorphosis. Colonies that came from larger larvae survived better, grew faster, and reproduced sooner or produced more embryos than colonies that came from smaller larvae. These effects crossed generations, with colonies from larger larvae themselves producing larger larvae. These effects were found in two populations (in Australia and in the United States) in contrasting habitats.

When the going gets rough: effect of maternal size manipulation on larval quality

Variation in larval size has been shown to be an important factor for the post-metamorphic performance of marine invertebrates but, despite its importance, few sources of this variation have been identified. For a range of taxa, offspring size is positively correlated with maternal size but the reasons for this correlation remain unclear. We halved the size of colonies in the bryozoan Bugula neritina 1 wk prior to reproduction (but during embryogenesis) to determine if larval size is a fixed or plastic trait. We manipulated colonies in such a way that the ratio of feeding zooids to reproductive zooids was constant between treatment and control colonies. We found that manipulating colony size strongly affects larval size; halved colonies produced larvae that were similar to13% smaller than those produced by intact colonies. We entered these data into a simple model based on previous work to estimate the likely post-metamorphic consequences of this reduction in larval size. The model predicted that larvae that came from manipulated colonies would suffer similar to300% higher post-metamorphic mortality and similar to50% lower fecundity as adults. Colonies that are faced with a stress appear to be trading off current offspring fitness to maximize their own long-term fitness and this may explain previous observations of compensatory growth in colonial organisms. This study demonstrates that larval size is a surprisingly dynamic trait and strong links exist between the maternal phenotype and the fitness of the offspring. The performance of settling larvae may be determined not only by their larval experience but also by the experience of their mothers.

Comparative reproductive biology of two southwestern Atlantic populations of the hermit crab Pagurus exilis (Crustacea: Anomura: Paguridae)

Pagurus exilis (Benedict, 1892) is an endemic South Atlantic hermit crab with a distribution ranging from Rio de Janeiro State, Brazil, to Buenos Aires Province, Argentina. The present study analyzed the reproduction of two populations at the extremes of this geographical distribution, and compared their reproductive period, fecundity, and changes in egg size and egg volume during the incubation period in order to assess the variability over this latitudinal range. Hermit crabs were collected monthly over a 2-year period. In total, 108 females were analyzed for Brazil (44 non-ovigerous and 64 ovigerous), and 141 for Argentina (87 non-ovigerous and 54 ovigerous). Reproduction in Brazil occurs year-round, with peaks in the fall and winter seasons; in Argentina reproduction occurs only in spring and summer. The Brazilian ovigerous females were significantly larger than the Argentina ones (Brazil: SL = 5.33 +/- 1.45 mm; Argentina: SL = 4.15 +/- 0.52 mm; P < 0.001). The fecundity was 1447 +/- 831 eggs (317 to 2885) in Brazil and 987 +/- 711 eggs (114 to 2665) in Argentina, with a trend towards higher fecundity in Brazil. Eggs in the Argentina population were larger than those in Brazil for all the three stages investigated, and no changes in egg volume were found during egg development for both populations. The reproductive traits of the two populations showed some important differences, which may reflect adaptations to local environmental conditions, demonstrating a high plasticity of reproductive features of the species in Brazilian and Argentine waters. The strategy adopted by the Argentina population involves a lower production of larger eggs compared to the population in Brazil; this lower production is associated with reproduction in cold-water regions.

Organization, evolution and transcriptional profile of hexamerin genes of the parasitic wasp Nasonia vitripennis (Hymenoptera: Pteromalidae)

Hexamerins and prophenoloxidases (PPOs) proteins are members of the arthropod-haemocyanin superfamily. In contrast to haemocyanin and PPO, hexamerins do not bind oxygen, but mainly play a role as storage proteins that supply amino acids for insect metamorphosis. We identified seven genes encoding hexamerins, three encoding PPOs, and one hexamerin pseudogene in the genome of the parasitoid wasp Nasonia vitripennis. A phylogenetic analysis of hexamerins and PPOs from this wasp and related proteins from other insect orders suggests an essentially order-specific radiation of hexamerins. Temporal and spatial transcriptional profiles of N. vitripennis hexamerins suggest that they have physiological functions other than metamorphosis, which are arguably coupled with its lifestyle.

Females of the weevil Zabrotes subfasciatus manipulate the size and number of eggs according to the host seed availability

In some insects, the finding of oviposition substrate triggers the uptake into oocytes of yolk proteins that are stored in the fat body during post-embryonic development. The main host of the bean weevil Zabrotes subfasciatus (Coleoptera; Chrysomelidae; Bruchinae; Amblycerini), in which larval resources are the sole source for future egg maturation, is Phaseolus vulgaris. Despite not feeding as adults, females of this species are able to lay eggs after encountering host seeds but it is not known how females react to changes in the availability of bean seeds. In the present study, the behaviour of Z. subfasciatus facing two very different environments for oviposition is investigated, as well as how this influences offspring fitness. The results obtained show that females of Z. subfasciatus react to variations in the availability of seeds belonging to the same host species by adjusting egg size and number. Females on low bean seed density lay larger and fewer eggs than those on high bean seed density, demonstrating a trade-off between these reproductive traits. Moreover, females can adjust egg size to changing levels of host availability during the first 4 days of their oviposition period. Although no difference in offspring weight is found, those from small eggs (low competition environment) result in larger adults. No response to selection on these traits after rearing beetles on the same host for 40 generations is observed. This unresponsiveness may indicate that beetle populations behave according to their reaction norm that already allows rapid adaptation to a varying amount of host-seed availability and better exploitation of the environments of this widespread stored-seed pest.

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPQ were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes. (C) 2008 Elsevier B.V. All rights reserved.

Variable effects of larval size on post-metamorphic performance in the field

Larval quality may be capable of explaining much of the variation in the recruitment and subsequent population dynamics of benthic marine invertebrates. Whilst the effects of larval nutritional condition on adult performance have received the most attention, recent work has shown that larval size may also be an important and ubiquitous source of variation in larval quality. We examined the effects of variation in larval size on the post-metamorphic survival and growth of Watersipora subtorquata in 2 very different habitats - experimental substrata and pier pilings. We found strong effects of larval size on colony performance, although these varied among experiments. For colonies on experimental substrata, larval size positively affected adult survival and, initially, growth. However, after 3 wk in the field, there was no relationship between larval size and colony size, possibly because colonies were completely surrounded by newly settled organisms. Larval size also positively affected post-metamorphic growth of colonies on pier pilings, but, surprisingly, colonies that came from larger larvae had lower survival than colonies from smaller larvae. Overall, variation in larval size will strongly affect the recruitment and subsequent performance of adults in this species, although this may vary among different habitats. This study highlights the importance of examining the effects of larval quality on adult performance in as realistic conditions as possible, because of the strong interaction between larval size effects and the environment.

Surface miscibility of EPC/DOTAP/DOPE in binary and ternary mixed monolayers

Surface pressure (pi)-molecular area (A) curves were used to characterize the packing of pseudo-ternary mixed Langmuir monolayers of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and L-alpha-dioleoyl phosphatidylethanolamine (DOPE). This pseudo-ternary mixture EPC/DOPE/DOTAP has been successfully employed in liposome formulations designed for DNA non-viral vectors. Pseudo-binary mixtures were also studied as a control. Miscibility behavior was inferred from pi-A curves applying the additivity rule by calculating the excess free energy of mixture (Delta G(Exc)). The interaction between the lipids was also deduced from the surface compressional modulus (C(s)(-1)). The deviation from ideality shows dependence on the lipid polar head type and monolayer composition. For lower DOPE concentrations, the forces are predominantly attractive. However, if the monolayer is DOPE rich, the DOTAP presence disturbs the PE-PE intermolecular interaction and the net interaction is then repulsive. The ternary monolayer EPC/DOPE/DOTAP presented itself in two configurations, modulated by the DOPE content, in a similar behavior to the DOPE/DOTAP monolayers. These results contribute to the understanding of the lipid interactions and packing in self-assembled systems associated with the in vitro and in vivo stability of liposomes. (C) 2010 Elsevier B.V. All rights reserved.