Relative migration rates and local adaptation in a mosquito-protozoan interaction.

Theory predicts that the direction of local adaptation depends on the relative migration rates of hosts and parasites. Here we measured relative migration rates and tested for local adaptation in the interaction between a tree hole mosquito (Ochlerotatus sierrensis) and a protozoan parasite (Lambornella clarki). We found strong support for the hypothesis that the host migrates more than its parasite. Hosts colonized artificial tree holes in the field at a much higher rate than the parasite. Field releases of the parasite demonstrated that it colonizes and persists in natural tree holes where it was previously absent, suggesting that parasite distribution is limited by its migratory ability. Although the host migrates more than its parasite, we found no evidence for local adaptation by hosts and some evidence for local adaptation by parasites. Other life history traits of the host and parasite may also influence patterns in local adaptation, particularly parasite virulence and host dormancy.

Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea.

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV.

Co-infection of Manduca sexta larvae with polydnavirus from Cotesia congregata increases susceptibility to fatal infection by Autographa californica M Nucleopolyhedrovirus.

We investigated pathogenesis of Autographa californica M Nucleopolyhedrovirus in the semipermissive host, Manduca sexta, using a lacZ recombinant virus (AcMNPV-hsp70/lacZ) to track the temporal progression of infection. Results from time course studies monitoring infections initiated orally in fourth instars demonstrated that primary infection of midgut columnar cells began at 3 h post inoculation (hpi). We observed secondary infections in midgut-associated tracheae as early as 9 hpi, showing that the early events of pathogenesis in M. sexta are similar to those of permissive noctuid larvae. In M. sexta, however, unlike in permissive hosts, hemocytes rapidly surrounded infected tracheal cells and formed capsules. Subsequently, baculovirus infections failed to spread and ultimately were cleared, suggesting that a cellular immune response had been triggered. To assess the effects of immunosuppression on baculovirus-induced disease, we compared the outcome of infections in immunocompetent hosts with those that were immunocompromised either by parasitization with the braconid, Cotesia congregata, or by injection of the parasitoid's polydnavirus. During the first 9 days after inoculation, parasitized and polydnavirus-inoculated M. sexta larvae died more quickly and at higher levels than nonparasitized and sham-injected controls, suggesting that the cellular immune response was a factor in conferring resistance to fatal infection by AcMNPV-hsp70/lacZ.

Multiple nucleocapsid packaging of Autographa californica nucleopolyhedrovirus accelerates the onset of systemic infection in Trichoplusia ni.

Among the nucleopolyhedroviruses (Baculoviridae), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) containing a beta-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar Trichoplusia ni were challenged with doses of ODV-S or ODV-M that yielded the same final mortality ( approximately 70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of AcMNPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.

AcMNPV pathogenesis and developmental resistance in fifth instar Heliothis virescens.

Autographa californica M nucleopolyhedrovirus carrying a lacZ reporter gene was used to study pathogenesis and developmental resistance in fifth instar Heliothis virescens. Compared to fourth instar larvae, the progression of infection proceeded much more slowly in fifth instar larvae, and developmental resistance in orally inoculated larvae was much more sudden and severe. The resistance occurred within the first 6 h of the fifth instar and was partially relieved by the optical brightener M2R. Inclusion of M2R in oral inocula not only increased mortality levels, but also increased both the percentages of insects expressing lacZ and the numbers of foci 24 h after infection. This early developmental resistance was not observed in insects infected intrahemocoelically, indicating that it was mediated by the midgut and/or the tracheal system servicing the midgut. Another less severe resistance was encountered in insects intrahemocoelically infected 36 to 48 h postmolt. This later resistance was partially relieved by methoprene indicating that it was, in part, hormonally mediated. Fifth instar insects fatally infected prior to 12 h postmolt never pupated, but pupation increasingly occurred in insects fatally infected 12 to 48 h postmolt. This pattern of larval/pupal death was consistent with viral inactivation of the ecdysone commitment peak.

Regulatory factors affecting larval mosquito populations in container and pool habitats: implications for biological control.

Empirical studies have shown that populations of larval mosquitoes developing in ground pools are subjected to different mortality factors than populations developing in water-filled containers. In general, larvae in ground pools are limited by natural enemies whereas those in containers are limited by resource availability. Containers also are typically smaller than ground pools and lack appreciable internal primary productivity. These physical and biological features have significant implications for successful implementation of biological control agents. Island biogeography theory suggests that container habitats will support smaller populations of fewer species compared to ground pools, implying that it may be more difficult to establish natural enemies in container habitats. The lack of primary productivity within containers may limit the number of trophic levels and reduce the likelihood of establishing and maintaining predator populations. Most importantly, larval mosquito populations in containers are regulated by competitive interactions, and mortality from natural enemies is likely to be compensatory. These habitat and population characteristics, combined with difficulties in locating and treating containers, suggest that successful control of ground pool mosquitoes using biological control agents is more feasible.

Comparative pathogenesis of Autographa californica M nuclear polyhedrosis virus in larvae of Trichoplusia ni and Heliothis virescens.

We compared early viral pathogenesis and dose-mortality relationships for larvae of two highly susceptible hosts, Trichoplusia ni and Heliothis virescens, using a construct of AcMNPV containing the lacZ reporter gene. Larvae were inoculated either as newly molted fourth instars (4(0)) or 15 hr after the molt (4(15)). In 4(0)-inoculated larvae, first lacZ expression was detected in the midgut epithelium of T. ni at 4 hr postinoculation (hpi) compared to 18 hpi in H. virescens, and systemic infections were initiated from tracheole cells servicing the midgut epithelia beginning at 12 and 20 hpi, respectively. The longer viral tenure within the midgut and the slower progression of systemic infections within H. virescens ultimately contributed to its longer time to death. For 4(0)-inoculated H. virescens, proportions of lacZ-expressing larvae increased from 18 hpi until the onset of the molt to the fifth instar at 36 hpi; at this time point, the proportion of signaling insects equaled the final larval mortality. Viral infections within the midgut epithelium of H. virescens were lost during the molt to the fifth instar. Dose-mortality relationships suggested that the peritrophic membrane provided little protection from AcMNPV infections for either species.

Primary infection of insect tracheae by Autographa californica M nuclear polyhedrosis virus.

Previously we found that Autographa californica M nuclear polyhedrosis virus infections initiated in the midguts of insect hosts are disseminated within the hosts via tracheae, elements composing the insect respiratory system. Herein we report that infection of tracheae also can be initiated directly via the spiracles, exterior openings of the tracheal system to the atmosphere. Infections were initiated both by direct application of viral inoculum to the spiracles and by aerosol. Infections established by both these methods appeared to advance much more rapidly than infections established by the oral route, and a host highly refractory to infection orally was easily susceptible to direct tracheal infection. These findings are important for continuing efforts to improve the use of baculoviruses as pest control agents; they also establish the possibility that direct tracheal infection via aerosol may facilitate horizontal transmission of baculoviruses during natural epizootics.

The insect tracheal system: a conduit for the systemic spread of Autographa californica M nuclear polyhedrosis virus.

Baculoviruses establish systemic infections within susceptible insect hosts, even though host tissues are surrounded by basal laminae, extracellular matrices that exclude particles smaller than these viruses. Using a recombinant Autographa californica M nuclear polyhedrosis virus containing a lacZ reporter gene under the control of a constitutive promoter, we followed the progression of infection in Trichoplusia ni larvae. We discovered that infection of the larval insect tracheal system (and not hemocytes, as thought previously) provides the major conduit for this virus to pass through basal laminae and to spread throughout the host. Tracheal epidermal cells, the only known cellular components of the tracheal system, share a common lymph system. Locally these cells contact one another by interdigitating cytoplasmic extensions called epidermal feet. These two features of the tracheal system are likely to facilitate the rapid systemic spread of the virus. The findings reported here have major implications for the fields of insect pathology and biological control and usher in an important consideration regarding host-range factors.

Habitat overflow, a source of larval mortality for Aedes sierrensis (Diptera: Culicidae).

In laboratory and field tests, larvae of the western tree hole mosquito, Aedes sierrensis (Ludlow), were flushed by rainfall from both glass containers and natural tree holes. After 7 d and an accumulated rainfall of 7.9 cm (3.1 in), between 0 and 327 Ae. sierrensis immatures were recovered in collection devices placed below four natural tree holes. Habitat overflow affected all larval stages and may be an important, but overlooked, density-independent source of larval mortality for mosquitoes developing in some water-filled containers.

Correlation of Aedes sierrensis captures at human sentinels with CO2-baited Fay-Prince and duplex cone traps.

The efficiency of the duplex cone and Fay-Prince traps for monitoring adult male and female Aedes sierrensis was evaluated at 3 field sites in California. The numbers of females captured by both types of traps were significantly correlated with human sentinel collections. The Fay-Prince trap captured more Ae. sierrensis females than the duplex cone trap and was a better tool for estimating female activity levels. There was no significant correlation between the number of males captured in Fay-Prince traps and at humans. Male numbers in duplex cone trap collections explained only 27% of the variation in the number of males collected at sentinels, suggesting that neither trap is a robust tool for estimating male activity around humans.

Could Aedes albopictus (Diptera: Culicidae) become established in California tree holes?

The ability of temperate zone-adapted Aedes albopictus (Skuse) to survive and complete development in California tree holes was evaluated in laboratory experiments that assessed development under simulated wet-season conditions, larval competition with Aedes sierrensis at different food levels, temporal survivorship of eggs stored under different humidities and temperatures, and suitability of larvae as hosts for the indigenous parasite Lambornella clarki. At all resource levels, Ae. albopictus completed development at temperatures similar to those in natural tree hole water in spring and early summer (> or = 16 degrees C), but not those during the rainy winter months (4-11 degrees C). In competition studies at 21 degrees C, the population performance (i.e., survivorship, pupation time, and adult size) of Ae. albopictus at all resource levels was as good or better when larvae developed with Ae. sierrensis compared with when reared with only conspecifics. Egg survivorship declined with increased storage time, increased temperature, and decreased humidity; > 55% of eggs hatched following 24-wk storage at 11 degrees C with relative humidities > 78%. In host suitability tests, parasitic theronts of L. clarki consistently attacked Ae. albopictus larvae at rates significantly lower than Ae. sierrensis. L. clarki that successfully invaded Ae. albopictus larvae failed to multiply and kill their hosts; thus, Ae. albopictus is not a suitable host for L. clarki. The protracted drying of most tree holes and low water temperatures during the rainy season will hinder but not preclude establishment of Ae. albopictus in California.

Regulatory role of parasites: impact on host population shifts with resource availability.

Effects of infections by the ciliate Lambornella clarki on larval populations of its mosquito host Aedes sierrensis were examined in laboratory and field studies. When host populations developed with sufficient food, mortality from parasites was additive and reduced the number of emerging mosquitoes. For food-limited populations, mortality was compensatory or depensatory; emerging adults were as or more abundant with higher average fitness than those from uninfected control populations. When nutrients were scarce, parasitic infections relaxed larval competition and increased per capita food by reducing host abundance. Food limitation altered larval feeding behavior, reducing horizontal transmission and subsequent mortality from parasitism.

Parasitism of newly-hatched Aedes sierrensis (Diptera: Culicidae) larvae by Lambornella clarki (Ciliophora: Tetrahymenidae) following habitat flooding.

Host-parasite interactions between Lambornella clarki (Ciliophora: Tetrahymenidae) and its natural host, Aedes sierrensis (Diptera: Culicidae), were studied in newly flooded treeholes in northern California between 1986 and 1989. First instar host larvae hatched within 1 to 4 hr of flooding, while free-living trophonts of L. clarki appeared between 7 and 24 hr. As early as 24 hr after flooding, ciliates initiated the first parasite cycle by forming cuticular cysts on first instar larvae; by 64 hr, cysts were observed on larvae collected from all positive holes during all years. While larvae with as many as 12 cysts were observed, most supported only 1 cyst, and successful infections were established by the entry of a single ciliate into the host's hemocoel. Among treehole populations, the proportion of larvae with L. clarki cysts ranged from 2 to 100% at 48 hr indicating that enzootics and epizootics develop rapidly in newly flooded treeholes. Average attack rates from all holes by year ranged between 17.0 and 44.4%. Ciliates began entering hosts 48 to 72 hr after flooding, but some larvae escaped parasitization by molting to the second instar before ciliates penetrated the cuticle. In some treeholes, opportunistic microorganisms entered larvae with the invading ciliates and killed both the host and parasite.

Emergence characteristics of Aedes sierrensis (Diptera: Culicidae) from California treeholes with particular reference to parasite loads.

Wing lengths, emergence times, and parasite loads were determined for 8,444 newly emerged adult Aedes sierrensis (Ludlow) collected from 17 treehole emergence traps during a sampling period of 149 d. Peak adult emergence occurred in late spring and early summer, but a small second cohort developed in treeholes retaining water through the dry season. Mosquito production was significantly and positively correlated with the maximum volumes of treeholes. Male and female wing lengths and eclosion times varied significantly among populations from different treeholes; within a population, these variables were highly correlated, suggesting that larvae of both sexes respond similarly to treehole conditions. Egg number (but not longevity) was correlated with adult size for laboratory-maintained females collected from emergence traps. These results suggest variation in the vector potential of adult populations produced from different treeholes. Newly emerged mosquitoes were infected by five parasite species that depend on adult hosts for their dispersal. The mermithid nematode Octomyomermis troglodytis Poinar and Sanders and the ciliate Tetrahymena sp. were very rare in adult populations. Gamontocysts of the ascogregarine Ascogregarina clarki Sanders and Poinar infected the Malpighian tubules of adults from six treeholes. Ectoparasitic larval water mites (near Euthyas) were found on 12% of all adults eclosing from eight treeholes and selectively parasitized female hosts over males. The ciliate Lambornella clarki Corliss and Coats was the most common parasite; it infected males and females from seven treeholes in equal proportions. L. clarki had a significant negative impact on adult populations in that 13% of all females from L. clarki-positive treeholes were infected with ciliates, and infected females are parasitically castrated.

Predator-induced trophic shift of a free-living ciliate: parasitism of mosquito larvae by their prey.

Larvae of the treehole mosquito, Aedes sierrensis, release a waterborne factor that induces morphogenesis of one of their prey, the tetrahymenid ciliate Lambornella clarki. Induced free-living trophonts of L. clarki undergo a synchronous response in which cells divide and transform into parasitic cells (theronts) that encyst on larval predators. Parasitic ciliates penetrate the cuticle, enter the hemocoel, and ultimately kill their predator-host. In nature, this trophic shift can lead to predator extinction and dramatic changes in microbial populations. Facultative parasitism by this polymorphic ciliate may have evolved as an antipredator strategy. The experimentally inducible parasitic response of L. clarki provides a novel model for studying cellular morphogenesis of ciliated protozoa.

Dispersal of the parasitic ciliate Lambornella clarki: implications for ciliates in the biological control of mosquitoes.

Lambornella clarki (Ciliophora: Tetrahymenidae), an endoparasite of Aedes sierrensis (Diptera: Culicidae), is dispersed by infected adult mosquitoes. Invasion of the ovaries induces parasitically castrated females to exhibit oviposition behavior and thereby actively disperse ciliates through deposition into water. Oviposition behavior of infected females is prolonged and mimics that of normal gravid females in their first gonotropic cycle. Adults of both sexes also passively disperse ciliates by dying on water surfaces, and infected adults are more likely to die on water than uninfected adults. Ciliates dispersed by infected adults can infect larvae and form desiccation-resistant cysts. Parasite-induced dispersal by hosts, desiccation-resistant cysts, an active host-seeking infective stage, and high infection and mortality rates all indicate significant biological control potential for these and related ciliates against container-breeding mosquitoes.