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Keywords:

  • Aedes aegypti;
  • Aedes albopictus;
  • Culex quinquefasciatus;
  • Edhazardia aedis;
  • Vavraia culicis;
  • CuniNPV;
  • oviposition

ABSTRACT:

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

The impact of the presence of larval mosquito pathogens with potential for biological control on oviposition choice was evaluated for three mosquito species/pathogen pairs present in Florida. These included Aedes aegypti infected with Edhazardia aedis, Aedes albopictus infected with Vavraia culicis, and Culex quinquefasciatus infected with Culex nigripalpus nucleopolyhedrovirus (CuniNPV). Two-choice oviposition bioassays were performed on each host and pathogen species with one oviposition cup containing infected larvae and the other cup containing uninfected larvae (control). Both uninfected and E. aedis-infected female Ae. aegypti laid significantly fewer eggs in oviposition cups containing infected larvae. Uninfected gravid female Ae. albopictus and Cx. quinquefasciatus oviposited equally in cups containing uninfected larvae or containing larvae infected with V. culicis or CuniNPV, respectively. Gravid female Ae. albopictus infected with V. culicis did not display ovarian development and did not lay eggs. The decreased oviposition by gravid Ae. aegypti in containers containing E. aedis-infected larvae may indicate that the infected larvae produce chemicals deterring oviposition.


INTRODUCTION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Potential oviposition sites are located and evaluated by gravid mosquitoes using a combination of olfactory and visual cues (Bentley and Day 1989). In addition to abiotic factors, biotic factors are important in acceptability of a potential oviposition substrate (Clements 1999). Some mosquito species may be attracted to and oviposit in water containing oviposition pheromones released by previous females, or by the presence of eggs (Mboera et al. 2000, Braks et al. 2007), conspecific larvae (Wilmont et al. 1987, Zahiri and Rau 1998, Allan et al. 2005, Wachira et al. 2010), internal symbiotes Candida near pseudoglaebosa and Ascogregarina taiwanensis (Reeves 2004), or organic matter and associated microbes (Bentley and Day 1989, Trexler et al. 2003, Sumba et al. 2004). Avoidance of oviposition sites may occur in the presence of predators (Blaustein et al. 2004, 2005), entomopathogenic Bacillus spp. (Zahiri and Mulla 2005), larvae infected with Plagiorchis elegans (Rudolphi) (Zahiri et al. 1997), or even larvae under stress from over-crowding or starvation (Zahiri and Rau 1998). Chemically-mediated cues may be detected through antennal, tarsal, or gustatory receptors (Hudson 1956, Lee and Craig 1983, Bentley and Day 1989, Davis and Bowen 1994). Clearly, gravid mosquitoes are highly sensitive to many cues from potential oviposition sites; however, relatively little is known about the response of gravid females to the presence of many of the specific pathogens that imperil larval survival yet require larvae for the maintenance of the pathogen. Avoiding habitats that could potentially cause larval mortality is beneficial to the mosquito but detrimental to the pathogen, whereas the inability to detect a pathogenic environment benefits the pathogen but is detrimental to the mosquito.

Three mosquito species, important vectors of human and animal diseases in Florida, with associated larval pathogens were selected for evaluation of oviposition responses to substrates containing these mosquito pathogens. These included Ae. aegypti and Edhazardia aedis (Kudo), Aedes albopictus Skuse and Vavraia culicis (Weiser), and Culex quinquefasciatus Say and Culex nigripalpus nucleopolyhedrovirus (CuniNPV). The highly complex microsporidian pathogen E. aedis is species-specific to Ae. aegypti and spreads transovarially as well as horizontally when larvae consume the remains of dead, vertically-infected larvae. While most larvae with high infection rates die before metamorphosis, low to moderate infection rates appear to allow for development to adulthood (Becnel and Andreadis 1999). Microsporidia can be potentially successful biological control agents due to high host-specificity, safety for non-target organisms, and an ability to transmit vertically, which disperses the pathogen in the environment, and horizontally, which amplifies the pathogen (Becnel 1992, Becnel and Johnson 2000). Because oviposition is critical for dissemination and propagation of this transovarial pathogen species, understanding the impact of infection of ovipositing females as well as infection of larvae inhabiting oviposition sites on oviposition behavior is critical in our understanding of E. aedis transmission and its potential for use as a biological control agent.

Another microsporidian pathogen, V. culicis, is associated with 13 mosquito species across five genera (Becnel et al. 2005) and in Florida is most commonly detected in Ae. albopictus (Fukuda et al. 1997). Unlike E. aedis, V. culicis has a simple life cycle and host relationship, with the maintenance of the pathogen through horizontal transmission between larval hosts upon the death and ingestion of infected larvae (Becnel and Andreadis 1999, Kelly et al. 1981). Due to the fitness costs associated with infection, pupation levels can be low, particularly if larval nutrition is limited (Riviero et al. 2007). Maintenance of the pathogen relies heavily on continued oviposition by host species of mosquitoes in habitats containing the pathogen so that the pathogen has a continuous availability of larval hosts.

The baculovirus CuniNPV is present in wild populations of Cx. quinquefasciatus and Culex nigripalpus in Florida, and nearly all southeastern Culex with the exception of Cx. territans Walker are susceptible (Andreadis et al. 2003). Baculoviruses are highly virulent and generally uncommon in wild mosquito populations (Becnel et al. 2001). Infected larvae become behaviorally lethargic and remain at the water surface despite surrounding disturbances (Moser et al. 2001). Although CuniNPV more commonly infects larvae, the virus also has been isolated from adult mosquito midguts (Becnel et al. 2003). During the larval stages, the virus is spread by horizontal transmission when infected larvae die and release occlusion bodies containing infectious virions. If the mosquito survives to adulthood, the occlusion bodies are generally shed with the meconium shortly after emergence. Transmission can also occur when a newly emerged infected adult dies in a new aquatic habitat (Becnel et al. 2003). All three pathogens are highly virulent to their hosts, safe for non-target organisms, and have multiple modes of transmission, making them good candidates for biological control agents of their host mosquitoes.

The objective of this research was to examine the effect of pathogen presence in ovipositing females as well as pathogen presence in larvae residing in oviposition sites on oviposition site choice by Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus and associated specific pathogens, E. aedis, V. culicis, and CuniNPV, respectively.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Mosquitoes

Mosquitoes were laboratory-reared using methods described previously for Aedes (Allan and Kline 1998) and Cx. quinquefasciatus (Vrzal et al. 2010). Larvae of Ae. aegypti collected from Tampa, FL in 2008 were used to establish the Ae. aegypti colony. The colony of Ae. albopictus was established from a 2007 field collection from Gainesville, FL, and the colony Culex quinquefasciatus was established in 1995 from field collections from Gainesville, FL. Both Aedes species were blood fed on manually defibrinated bovine blood presented in collagen sausage casings (The Sausage Maker Inc., Buffalo, NY) and Cx. quinquefasciatus fed on chickens (University of Florida IACUC No. 469). Adults were maintained on a 5% sucrose solution in chambers at 28° C, 80 – 85% relative humidity, and a 14:10 h L:D photoperiod.

Pathogen maintenance and mosquito infection

These studies were conducted with three mosquito species with associated pathogens (Table 1) and required maintenance of pathogens and infected mosquitoes. All pathogens were obtained from J. Becnel, CMAVE/USDA, Gainesville, FL.

Table 1.  Experimental design with different mosquito and pathogen species. Thumbnail image of

A colony of Edhazardia aedis-infected Ae. aegypti was maintained using a modified protocol from Becnel and Undeen (1992). As this pathogen is transovarially-transmitted, infected Ae. aegypti larvae were obtained from colony eggs. To produce synchronized egg hatch, eggs were placed in deionized water and placed under vacuum for 10 min, with the vacuum turned off and the vacuum maintained for 10 additional minutes. After hatching (24 h), 100 larvae were transferred into each plastic cup (each containing 100 ml of water) with larval food slurry (0.5 ml) added. Larval food slurry consisted of 10 g of a 1:1 mixture of liver powder and brewer's yeast (MP Biomedical, Solon, OH) added to 100 ml of water. Every other day, 1 ml of larval food slurry was added to cups containing larvae until the 3rd instar. Samples of larvae were examined for uninucleate spores in the fat body by squashing larvae in a drop of deionized water on a slide and observation with phase-contrast at 400x magnification.

Vavraia culicis floridensis was maintained between Anopheles quadrimaculatus Say and Helicoverpa zea Boddie, 1850 as hosts to maintain infectivity in mosquitoes (Vavra and Becnel 2007). The pathogen was maintained following the protocol of Vavra and Becnel (2007). Approximately 400 uninfected Ae. albopictus larvae were hatched under vacuum. After 24 h, larvae were divided into two groups, and each group was placed in deionized water (100 ml) in petri dishes with only one larval dish receiving 0.25 ml purified V. culicis spores (1×104–5 spores/ml). The other dish (control) did not receive spores. Both dishes received 0.1 ml of larval food slurry. After 24 h, larvae were transferred to 30 × 18 × 5 cm ceramic pans containing 1 liter of well water. Larvae were fed 8 ml of larval food slurry every other day and reared to adulthood. Adults were scored for infection (presence or absence of spores) by randomly selecting individual adults, squashing under a cover slip in a drop of deionized water on a slide, and viewing at 40x magnification.

Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was supplied by J. Becnel at CMAVE/USDA laboratory in Gainesville, FL, and Cx. quinquefasciatus larvae were infected as described by Moser et al. (2001). Infected Cx. nigripalpus mosquitoes were originally field-collected in 1997 from a swine wastewater site in Gainesville, FL. CuniNPV field isolates were amplified in the laboratory using Cx. quinquefasciatus and virus collected and stored at −80° C in a solution containing 5×107 to 2×108 occlusion bodies/ml and 15 mM MgSO4. Groups of 50 infected larvae were frozen in deionized water at −80° C and used for culture maintenance. Approximately 3,000 uninfected early 3rd instar Cx. quinquefasciatus larvae were exposed to 100 larval equivalents of virus in solution with 14 mM MgCl2, and used for bioassays 48 h post-infection. Larvae were fed with 2 g dry larval diet per tray (∼0.7 μg /larva) at the time of exposure. Infected larvae, determined by levels of lethargy, generally do not survive to adulthood (Becnel et al. 2003).

Dual-choice oviposition bioassay methods

Initial assays were conducted using uninfected Ae. aegypti females following methods of Allan and Kline (1998). Forty-eight hours post-bloodfeeding, 10 gravid, nulliparous females were placed in a 30 × 30 × 30 cm screened bioassay cage. Each cage held two black 100 ml oviposition cups (Solo Cup Company, Highland Park, IL) containing 50 ml well water and placed in the center of the cage about 20 cm apart. Each oviposition cup contained one 8 × 6 cm strip of seed germination paper (Anchor Paper Company, St. Paul, MN) with striations oriented vertically as an oviposition substrate. Cups were designated as right and left and position noted for all assays to verify that there was no positional bias.

For the treatment cages, one oviposition cup contained ten uninfected 3rd instar Ae. aegypti larvae and the other cup contained ten E. aedis-infected 3rd instar larvae. Larvae were placed in oviposition cups immediately preceding the start of the bioassay. Control assay cages held two oviposition cups each containing ten uninfected 3rd instar larvae.

Females were allowed 24 h to oviposit with a 16:8 L:D photoperiod. Mosquitoes were not provided with sugar solution during the bioassays. After 24 h, oviposition papers were removed from cages and eggs and proportion of eggs in the treatment and control cups were calculated. Bioassays were repeated on at least three different days for a total of 30 treatment and control assays. Each day included treatment cages and control cages. Cages in which females did not lay eggs were excluded from the analysis.

Bioassays with infected female Ae. aegypti were conducted with individual mosquitoes following the same protocol as above. As it was not possible to obtain infection in 100% of females, assays were conducted using individual females so that infection could be verified for each assay. After bioassays were complete, females were examined to verify E. aedis infection. Any assays with uninfected females were not included in the analysis. A total of 30 treatment and control assays were completed.

Bioassays with Ae. albopictus were performed using uninfected Ae. albopictus larvae, as well as V. culicis-infected larvae as treatments. Although V. culicis-infected larvae may survive to adulthood (Riviero et al. 2007), infection appears to greatly reduce fecundity. Due to problems obtaining oviposition from infected adults, ovaries were dissected from a total of 45 infected adult females to check fecundity. Ovaries were dissected two, three, and four days after blood feeding, and egg development graded following Christophers' scale (Christophers 1911). Eggs developing past stage I were not found in infected females, and therefore oviposition choice bioassays evaluating infected gravid females could not be conducted.

Bioassays with Cx. quinquefasciatus were performed using ten uninfected Cx. quinquefasciatus larvae and ten live CuniNPV-infected Cx. quinquefasciatus larvae at 48 h post-inoculation as treatments. Two ml of hay infusion prepared following Reiter et al. (1991) was added to each oviposition cup to stimulate oviposition. Egg-rafts were counted, and the proportion of total egg-raft oviposition determined for each substrate. Preliminary evaluations indicated that egg-rafts did not differ in size. A total of 30 treatment and control assays were completed. Because CuniNPV-infected adults have low survival rates, infected adults were not included in this study.

Data were presented as the percentage of eggs or rafts in each cup of the total laid in each cage as this compensates for possible differences between cohorts of mosquitoes. Data within each comparison collected on different days were compared were tested for normality (Shapiro-Wilk test, α= 0.05) and compared by paired t-tests using SigmaStat 11.0 (Systat Software, Chicago, IL). As there were no differences between days, data were combined for each treatment. Data for each paired comparison were analyzed using SigmaStat 11.0 (Systat Software, Chicago, IL) and tested for normality (Shapiro-Wilk test, α= 0.05). If data passed normality testing, means were compared using paired t-tests. Data that did not pass normality were analyzed using the Mann-Whitney Rank Sum test.

RESULTS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

In preliminary dual-choice assays with uninfected female Ae. aegypti, there was no significant difference in the percent of eggs laid in either oviposition cup containing uninfected larvae, indicating a lack of positional bias in the cage (49.9 ± 1.9%, 50.1 ± 1.9%) (t= 0.08; df = 58; P= 0.93). However, when exposed to oviposition cups containing uninfected larvae or E. aedis-infected larvae, uninfected Ae. aegypti females laid significantly fewer eggs in oviposition cups containing infected larvae (Table 2).

Table 2.  Oviposition responses of gravid female Ae. aegypti (uninfected or infected with E. aedis), uninfected Ae. albopictus, and uninfected Cx. quinquefasciatus in dual-choice oviposition assays to cups containing either uninfected or pathogen-infected larvae (N = 30). Thumbnail image of

Edhazardia aedis-infected Ae. aegypti did not differentiate between the two oviposition cups containing uninfected larvae (52.1 ± 6.3%, 47.9 ± 6.3%) (U = 1.064; df = 30, 30; P= 0.609), again indicating a lack of positional bias. Female Ae. aegypti infected with E. aedis laid significantly fewer eggs in oviposition cups containing E. aedis-infected larvae than those containing uninfected containing larvae (Table 2). A comparison of the egg production by infected and uninfected Ae. aegypti females was done using data from control assays only as the presence of infected assays may have had an impact of overall egg production. In the presence of uninfected larvae (control assays), each uninfected Ae. aegypti female produced 34.2 ± 1.8 eggs and E. aedis-Ae. aegypti produced 18.0 ± 3.4 eggs. Infection with E. aedis reduced overall oviposition in assays (U = 994.5; df = 60, 60; P < 0.001).

In the preliminary control assays, uninfected Ae. albopictus females deposited eggs equally in the two control oviposition cups containing uninfected larvae (47.7 ± 4.3%, 52.3 ± 4.3%) (t= 0.772; df = 58; P= 0.438), indicating no positional bias for either side of the cages. Uninfected female Ae. albopictus showed no significant difference in the percent of eggs laid between oviposition cups containing uninfected larvae and oviposition cups containing V. culicis-infected larvae (Table 2). Numbers of eggs between treatments ranged from 37.3–43.8 eggs/cup.

There was no significant difference in the preliminary control assays in the percentage of egg rafts deposited by uninfected Cx. quinquefasciatus females in two control oviposition cups, each containing healthy larvae (45.6 ± 2.5%, 54.4 ± 2.5%)(t= 1.607; df = 58; P= 0.112), indicating no positional bias for either side of the cages. Female Cx. quinquefasciatus exhibited no significant difference in the percent of egg rafts distributed between oviposition cups containing uninfected larvae or CuniNPV-infected larvae (Table 2). Numbers of egg rafts for treatments were similar and ranged from 2.5–3.4 rafts per cup.

DISCUSSION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Previous studies have reported on avoidance by gravid female mosquitoes of potential oviposition sites that may jeopardize survival of their progeny. In particular, strong avoidance has been documented for water containing vertebrate predators such as fish (Angelon and Petranka 2002), amphibians (Blaustein and Kotler 1993, Mokany and Shine 2003), and invertebrate predators (Blaustein et al. 2004, 2005). While it remains unclear from these studies if avoidance was visually or chemically mediated, several studies have provided evidence of predator-released kairomones that deter mosquito oviposition (Angelon and Petranka 2002, Mokany and Shine 2003, Blaustein et al. 2004, 2005). In the case of notonectids, the hydrocarbons n-heneicosane and n-tricosane appear to be involved with mosquito oviposition deterrence (Silberbush et al. 2010). Similar suppression of mosquito oviposition behavior in response to parasites and pathogens has also been reported. Lowenberger and Rau (1994) observed reduced oviposition by Ae. aegypti females in the presence of larvae parasitized by P. elegans over a range of infection levels. This oviposition deterrence from parasitized larvae was not considered a reaction to an organic substance but rather a reaction to cues emitted by parasitized larvae (Lowenberger and Rau 1994). In laboratory oviposition studies, Ae. albopictus females oviposited more in water containing Bacillus thuringiensis var. israelensis (Bti), compared to control water, suggesting that females did detect Bti; however, under field conditions, no differences were detected (Stoops 2004). In contrast, oviposition levels by Cx. quinquefasciatus were inversely related to high concentrations of Bti and Bacillus sphaericus (Zahiri and Mulla 2005). Differences in the responses of Ae. albopictus and Cx. quinquefasciatus to Bti may relate to behavioral differences as Ae. albopictus females do not drink from the substrate as do Culex females, and thus Ae. albopictus may not detect chemicals from water with phagoreceptors.

In the current study, oviposition by both E. aedis-infected and uninfected gravid Ae. aegypti females was negatively impacted by the presence of conspecific larvae infected with the microsporidian pathogen, E. aedis. However, oviposition by gravid uninfected Ae. albopictus females or uninfected Cx. quinquefasciatus females was not impacted by the presence of conspecific larvae infected with the microsporidium V. culicis or the baculovirus CuniNPV, respectively. Gravid Ae. aegypti were able to detect the presence of E. aedis possibly because E. aedis is specific to Ae. aegypti and presumably the two organisms have co-evolved and currently exist in a dynamic equilibrium. Edhazardia aedis was the most widespread and prevalent pathogen of Ae. aegypti found in Thailand (Hembree 1979), but overall infection levels in these populations was not determined. At this point, one can only speculate on the observed deterrence of females from ovipositing in substrates with E. aedis-infected larvae. Female Ae. aegypti, regardless of infection status, may more often than not avoid larval habitats containing E. aedis. It is important to note that oviposition deterrence was not complete and some females laid eggs on the substrate with infected larvae, suggesting variation in the Ae. aegypti population for the ability to detect E. aedis-infected habitats. Edhazardia aedis is specific for Ae. aegypti with a likely complex and highly evolved relationship, which implies that a host-pathogen balance must be maintained, and the presence of variation in the host population for the ability to detect E. aedis, or the willingness to oviposit regardless of larval infection status, supports this. As a host-specific obligate pathogen, E. aedis requires the availability of populations of Ae. aegypti in order to survive, and one scenario that would create sustained host populations includes variation in susceptibility to the pathogen by host larvae, as well as variation in behavior of ovipositing females with regard to choosing larval habitat. The observed oviposition behavior of female Ae. aegypti may reflect this balance where the pathogen can persist and spread within a portion of the mosquito population while a portion of the population remains free of the pathogen.

There was no impact on oviposition by uninfected gravid Ae. albopictus and Cx. quinquefasciatus females in the presence of conspecific larvae infected with V. culicis or CuniNPV, respectively. CuniNPV and V. culicis and are very different pathogens, and neither is species specific. Vavraia culicis infects multiple mosquito genera, as well as multiple insect families, and it only develops within a single generation (larvae and adults) of the host (Vavra and Becnel 2007). CuniNPV infects many species of Culex mosquitoes but not species of other mosquito genera or any other insects (Andreadis et al. 2003). Species-specificity of a pathogen suggests specialization by the pathogen (Poulin and Mouillot 2003), which is not the case for V. culicis and CuniNPV.

The identity and sources of the substances that resulted in decreased oviposition by Ae. aegypti in our study remain unknown; however, chemical cues as a result of altered larval physiology or stress due to E. aedis-infected larvae may be involved. Alternatively, microbial fauna commonly associated with enhanced oviposition by gravid mosquitoes (Trexler et al. 2003) could be altered in infected larvae changing the composition of attractant cues in conspecific larvae to become neutral or repellant. Larvae of Ae. aegypti when starved or in overcrowded conditions can become stressed resulting in ovipositional deterrence by Ae. aegypti females (Zahiri and Rau 1998). Some pathogens, parasites, and predators clearly alter oviposition sites and deter mosquito oviposition. Further research should focus on the mechanisms of this deterrence from such relationships.

Acknowledgments

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

We thank Neil Sanscrainte, Erin Vrzal-Wessels, Haze Brown, and Julie McClurg for their technical support and guidance, and the University of Florida Entomology and Nematology Department for providing financial support through a graduate assistantship.

REFERENCES CITED

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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