Potential use of neem leaf slurry as a sustainable dry season management strategy to control the malaria vector Anopheles gambiae (DIPTERA: CULICIDAE) in west African villages.

Larval management of the malaria vector, Anopheles gambiae Giles s.s., has been successful in reducing disease transmission. However, pesticides are not affordable to farmers in remote villages in Mali, and in other material resource poor countries. Insect resistance to insecticides and nontarget toxicity pose additional problems. Neem (Azadirachta indica A. Juss) is a tree with many beneficial, insect bioactive compounds, such as azadirachtin. We tested the hypothesis that neem leaf slurry is a sustainable, natural product, anopheline larvicide. A field study conducted in Sanambele (Mali) in 2010 demonstrated neem leaf slurry can work with only the available tools and resources in the village. Laboratory bioassays were conducted with third instar An. gambiae and village methods were used to prepare the leaf slurry. Experimental concentration ranges were 1,061-21,224 mg/L pulverized neem leaves in distilled water. The 50 and 90% lethal concentrations at 72 h were 8,825 mg/L and 15,212 mg/L, respectively. LC concentrations were higher than for other parts of the neem tree when compared with previous published studies because leaf slurry preparation was simplified by omitting removal of fibrous plant tissue. Using storytelling as a medium of knowledge transfer, villagers combined available resources to manage anopheline larvae. Preparation of neem leaf slurries is a sustainable approach which allows villagers to proactively reduce mosquito larval density within their community as part of an integrated management system.

Effects of starvation and parasitism on foregut contraction in larval Manduca sexta.

Larvae of Manduca sexta are parasitised by the braconid wasp, Cotesia congregata. In this study we examined whether contraction activity of the semi-isolated foregut was affected by parasitism. Parasitised larvae fed significantly less compared with unparasitised control larvae, therefore starved unparasitised animals were used as controls. Rate and force of foregut contraction in control caterpillars significantly increased with days of starvation. However, only contraction force in foreguts of parasitised larvae increased over time following infection. The presence of food in the foregut of caterpillars starved 7 days suggested that food moved anteriorly from the midgut and that contraction became antiperistaltic, but only normal peristalsis occurred in parasitised caterpillars. Rate and force of gut contractions may be controlled independently and starvation did not truly mimic the effects of the parasitoids. Dissection of caterpillars with emerged wasps indicated that 47% had a single wasp larva wedged between the brain and foregut. Removal of this wasp caused an increased rate of foregut contraction of the caterpillar. Brain removal resulted in an increased rate of foregut contraction only for unparasitised insects. Sectioning of the recurrent nerve temporarily eliminated foregut contraction, but the contraction began again in 250 s in parasitised caterpillars prior to wasp emergence, compared with over 500 s for unparasitised controls and parasitised caterpillars following wasp emergence.

Effects of terrestrial pollutants on insect parasitoids.

Parasitoids are important organisms in the regulation of insect herbivores in natural, urban, and agricultural ecosystems. The impact of pollutants acting on parasitoids has not been extensively reviewed. This prompted us to propose a falsifiable null hypothesis (pollutants have no effects on parasitoids) and two alternative hypotheses (pollution negatively or positively affects parasitoids) to assess in the available literature the effects of pollutants acting on parasitoids. We found 26 studies examining 39 biological systems that met our criteria for inclusion. Of these studies, 18 of the 39 biological systems (46.2%) supported the null hypothesis while 18 (46.2%) supported the first alternative hypothesis in which pollutants exhibited negative effects on parasitoids. Only a small percentage of the studies (7.6%, 3 of 39) supported the second alternative hypothesis suggesting that pollutants had positive effects on parasitoids. We provide a synthesis of the available data by pollution type, summarize trends for different pollutants, and suggest future areas of research.

Comparison of three methods of parasitoid polydnavirus genomic DNA isolation to facilitate polydnavirus genomic sequencing.

A major long-term goal of polydnavirus (PDV) genome research is to identify novel virally encoded molecules that may serve as biopesticides to target insect pests that threaten agriculture and human health. As PDV viral replication in cell culture in vitro has not yet been achieved, several thousands of wasps must be dissected to yield enough viral DNA from the adult ovaries to carry out PDV genomic sequencing. This study compares three methods of PDV genomic DNA isolation for the PDV of Cotesia flavipes, which parasitizes the sugarcane borer, Diatraea saccharalis, preparatory to sequencing the C. flavipes bracovirus genome. Two of these protocols incorporate phenol-chloroform DNA extraction steps in the procedure and the third protocol uses a modified Qiagen DNA kit method to extract viral DNA. The latter method proved significantly less time-consuming and more cost-effective. Efforts are currently underway to bioengineer insect pathogenic viruses with PDV genes, so that their gene products will enhance baculovirus virulence for agricultural insect pests, either via suppression of the immune system of the host or by PDV-mediated induction of its developmental arrest. Sequencing a growing number of complete PDV genomes will enhance those efforts, which will be facilitated by the study reported here.

[Co-evolutionary strategies of interaction between parasitoids and polydnaviruses]. / Estrategias co-evolutivas de la interacción entre parasitoides y polidnavirus.

Polydnaviruses are genetic symbionts of wasp endoparasitoids belonging to the hymenopteran families Ichneumonidae (ichnoviruses) and Braconidae (bracoviruses). They exist as proviruses integrated in the wasp's chromosomal genome, which then excise and undergo replication during the stage of adult development of the wasp. During wasp oviposition into their caterpillar host, the fully formed virus particles are injected along the parasitoid's eggs into the host hemocoel, where the eggs hatch and undergo larval development. The primary function of the polydnavirus is to trigger host immunosuppression so that host hemocytes are prevented from encapsulating the parasitoid's eggs and/or larvae. Polydnavirus transcripts are expressed following parasitization and alter host hemocyte adhesive properties that prevents encapsulation; in some species, viral gene expression triggers host hemocyte apoptosis, thereby rendering the host immunosuppressed. This review summarizes the major features of polydnaviruses and provides a global view of their functions in the lepidopteran hosts of the parasitoid wasps that carry them both as integrated viral sequences in their genome and as free virus to function physiologically in host regulation following parasitization of the host.

Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes.

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response.

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.

Comparative larvicidal toxicities of three ecdysone agonists on the mosquitoes Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae.

Ecdysone agonists are hormonally active insect growth regulators that disrupt development of pest insects and have potential for development as insecticides. Their effects have been particularly well-studied in Lepidoptera and Coleoptera, but significantly less is known about their effects on dipterans, particularly aquatic species. The potency of three ecdysone agonists on larvae of 3 mosquito species, Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus, was examined. Anopheles gambiae was the most susceptible species and Ae. aegypti was the most resistant species to the effects of the three compounds tested. Potency, in descending order, was RH-2485 > RH-5992 > RH-5849. Dose-response relationships were determined for the three agonists; RH-2485 was found to be the most effective endocrine disruptor against all three species. The observed biological effects of these compounds were similar to those reported for other insects, and mosquitoes initiated molting and apolysis but did not complete a molt. In some cases, mosquito larvae synthesized a new cuticle that appeared to be normally sclerotized but the larvae failed to ecdyse and shed the exuvium. These compounds may prove to be valuable insect growth regulators for control of mosquitoes to decrease the frequency of pathogen transmission to humans. Prospects for using these compounds to control mosquitoes in the field are discussed, along with possible impacts on non-target arthropods in mosquito habitats.

Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control.

Wasp parasitoids use a variety of methods to commandeer their insect hosts in order to create an environment that will support and promote their own development, usually to the detriment of the host insect. Parasitized insects typically undergo developmental arrest and die sometime after the parasitoid has become independent of its host. Parasitoids can deactivate their host's immune system and effect changes in host hormone titers and behavior. Often, host tissues or organs become refractory to stimulation by tropic hormones. Here we present an overview of the manipulative capabilities of wasp-injected calyx fluid containing polydnaviruses and venom, as well as the parasitoid larva and the teratocytes that originate from the serosal membrane that surrounds the developing embryo of the parasitoid. Possibilities for using regulatory molecules produced by the parasitoid or its products that would be potentially useful in developing new, environmentally safe insect control agents are discussed.

Visualization of polydnavirus sequences in a parasitoid wasp chromosome.

Polydnaviruses, obligatorily associated with endoparasitoid wasps, are unique in that their segmented genome is composed of multiple double-stranded DNA circles. We present here the first cytological evidence that virus segments are integrated in the wasp genome, obtained by using in situ hybridization of virus probes with viral sequences in the chromosomes of a wasp from the braconid family of hymenopterans.

Inhibition of the larval ecdysis and emergence behavior of the parasitoid Cotesia congregata by methoprene.

Parasitism of the tobacco hornworm, Manduca sexta, by the braconid wasp Cotesia congregata, induces developmental arrest of the host in the larval stage. During the final instar of the host, its juvenile hormone (JH) titer is elevated, preventing host metamorphosis. This study investigated the effects of hormonal manipulation of the host on the parasitoid's emergence behavior. The second larval ecdysis of the wasps coincides with their emergence from the host, and application of the juvenile hormone analogue methoprene to day 4 fifth instar hosts either delayed or totally suppressed the subsequent emergence of the wasps. Effects of methoprene were dose-dependent and no parasitoids emerged following treatment of host larvae with doses >50 &mgr;g. Parasitoids which failed to emerge eventually succumbed as unecydsed pharate third instar larvae in the hemocoel of the host. Effects of host methoprene treatment on parasitoid metamorphosis were also assessed, and metamorphic disruption occurred at much lower dosages compared with doses necessary to suppress parasitoid emergence behavior. The inhibitory effect of methoprene on parasitoid emergence behavior appears to be mediated by effects of this hormone on the synthesis or release of ecdysis-triggering hormone (ETH) in the parasitoid, the proximate endocrine cue which triggers ecdysis behavior in free-living insects. ETH accumulated in the epitracheal Inka cells of parasitoids developing in methoprene-treated hosts, suggestive of a lack of hormone release. Thus, the hormonal modulation of parasitoid emergence behavior appears to be complex, involving a suite of hormones including JH, ecdysteroid, and peptide hormones.