The proposed expansion of biofuels production may cause unintended land-use changes and potentially alter ecosystem services. This study evaluated the impact of first-generation (corn) and second-generation (switchgrass and Miscanthus) biofuel crops on production and oviposition site selection by two vector mosquitoes, the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. Larvae of the two species were reared at varying conspecific and heterospecific densities in senescent leaf infusions prepared from one of the three biofuel crops and their survival and development time to adulthood determined. The effects of the three leaf infusions on water chemistry and oviposition site selection by the two mosquito species were also determined. Ae. albopictus females deposited significantly fewer eggs in Miscanthus than in corn infusion while Ae. aegypti females deposited significantly fewer eggs in Miscanthus than in both corn and switchgrass infusion. Survival to adulthood for both mosquito species was significantly lower in corn than in switchgrass and Miscanthus infusions; was consistently lower at high- (0:40 and 20:20) than at low density treatments in both switchgrass and Miscanthus infusions; and significantly lower at high intraspecific density (40:0 and 0: 40) than at high interspecific density (20:20) in Miscanthus infusion. Development time to adulthood was positively related to larval density, but was not influenced by biofuel leaf treatment. Corn infusion had lower pH values and higher salinity, conductivity, total dissolved solids (TDS), and temperature values than switchgrass and Miscanthus infusions. These findings demonstrate the potential for biofuel crops to modify the chemistry of aquatic habitats in ways that may influence mosquito production and thereby the risk of exposure to mosquito-borne diseases.
Due to the increasing economic and environmental costs of energy production from fossil fuels, many governments, including the U.S., seek increased energy production from biofuels derived from a variety of renewable feedstocks. Currently, the majority of U.S. biofuel production is in the form of ethanol derived from starch- or grain-based feedstocks, with over 90% of the biofuels produced and used domestically coming from corn (Groom et al., 2008). Because corn production requires more fertilizers and pesticides than any other major U.S. food or biofuel crop (USDA-NASS, 2007), and requires energy inputs that may limit the desired energetic benefits over fossil fuels (Vadas et al., 2008), there is increasing interest in biofuel crops that can produce high-energy yields per hectare under low-input methods. These so-called ‘second-generation’ biomass feedstocks, which include several perennial grasses, may offer economic and environmental advantages by requiring less energy, water, fertilizers, and pesticides, as well as increasing carbon sequestration and improving habitat for wildlife (Simmons et al., 2008). Among the leading candidates for second-generation biomass feedstocks are two perennial grasses, Miscanthus (Miscanthus x giganteus) and switchgrass (Panicum virgatum), which require fewer inputs and offer increased yields over first-generation biomass feedstocks such as corn (Graham et al., 1995; Parrish & Fike, 2005; Adler et al., 2007; Heaton et al., 2008; Vadas et al., 2008). Furthermore, recent efforts have also targeted the potential value of restored native prairie polycultures, which are particularly well-adapted to the climate and soils of the central U.S., require few inputs, and also offer a substantially higher conversion efficiency than corn (Tilman et al., 2006).
Despite the growing interest in second-generation biofuels as alternative sources of energy, there are concerns that land-use changes associated with their production may have detrimental environmental impacts. Conversion of forests and grasslands into biofuel crop lands could increase carbon emissions (Searchinger et al., 2008), and areas that have recently experienced increased levels of protection due to declining values of food crops are once again under consideration for biofuel production (Marshall 2007). There is further concern that biofuel production will decrease global food supplies by diverting resources necessary for food production, namely land, water, and energy, away from food production (Pimentel et al., 2009).
Although rarely studied, ecological changes associated with biofuel production have myriad potential to influence human well-being, such as through changes in the transmission dynamics of vector-borne diseases. Potential impacts of biofuel production on vector-borne diseases may occur via a variety of mechanistic pathways. First, transitions from one crop to another can create new breeding sites for mosquitoes and destroy others leading to elimination of some species and establishment of others (Buck et al., 1972; Amerasinghe & Munasingha, 1988; Amerasinghe & Indrajith, 1994). Second, crop transitions may alter the local microclimate (e.g., temperature, relative humidity) directly affecting vector survivorship, development, and blood feeding rates (Patz et al., 2000; Daszak et al., 2001). Third, ecological changes associated with crop transitions may also influence vector diversity and abundance by changing the availability and density of preferred vertebrate hosts (the blood meal sources). For example, the establishment of row crop agriculture has decreased the diversity of associated avian populations, while providing forage for large flocks of bird species that are highly competent reservoirs for West Nile Virus (WNV) (e.g., common grackle, American crow; (Kilpatrick, 2011)). Finally, the chemical and nutritional composition of surrounding aquatic habitats may be altered by inputs of senescent tissues of biofuel plants affecting their suitability as breeding sites for disease vectors such as mosquitoes.
While croplands occupy a considerable proportion of many states (the 21.45 million acres of Illinois corn and soybeans harvested in 2010 represented 60% of the State's total land area [USDA-NASS, 2011)], we know little about the impact of these familiar crops or proposed second-generation biofuel crops on the life history characteristics of mosquitoes that ultimately determine the dynamics of mosquito-borne diseases. Considering the present, wide distribution of these agricultural crops, and the potential transition to second-generation perennial grass biofuels, an improved understanding of the potential consequences of agricultural and biofuel production for human and wildlife health is needed.
The aim of the current study was to investigate the influence of first-generation (corn) and second-generation (switchgrass and Miscanthus) biofuel crops on mosquito survival, development, and oviposition site selection. Mosquitoes are vectors of infectious diseases to humans that are among the greatest public health threats (e.g. malaria, dengue, WNV, lymphatic filariasis) and thus are major targets of vector control. Adding to this challenge, mosquitoes exhibit a complex life cycle with aquatic immature stages (larvae and pupae) and a terrestrial adult stage. Immature stages of many species of mosquitoes thrive in aquatic habitats within and around the vicinity of agricultural lands that often receive large inputs of senescent leaf litter from surrounding cultivated crops. Because detritus type is known to influence mosquito production (Fish & Carpenter, 1982; Murrell & Juliano, 2008) and oviposition behavior (Ponnusamy et al., 2010), understanding the impact of biofuel crops on these processes will assist policy makers in determining the types of biofuel crops that increase energy production while balancing the potentially negative impacts of land-use change on disease risk. Here, we used two important vector species, the yellow fever mosquito Ae. aegypti and the Asian tiger mosquito Ae. albopictus, to test the hypothesis that biofuel crops have differential effects on mosquito production and oviposition site selection by gravid female mosquitoes.
Material and methods
The experiments described herein were conducted using F10 generation of Ae. aegypti and Ae. albopictus originally obtained from field collections in Florida. To maintain the colony, larvae of each species were reared in larval pans in batches of 100 individuals and fed on yeast + albumin (1 : 1); adults were maintained in plastic cages at 27 ± 1 °C and fed 20% sucrose. For egg production, the females were blood fed twice per week; once with a restrained guinea pig and once with artificial membrane feeding system (Hemotek, Lancaster, UK).
Conspecific larvae of Ae. aegypti and Ae. albopictus were reared in larval pans and emerging adults were maintained according to previously described methods (Muturi et al., 2011). Five- to ten-day-old adult females were provided 30 min access to citrated bovine blood by an artificial membrane feeding system (Hemotek, Lancashire, United Kingdom). Blood fed females of each species were housed in paperboard cages in groups of five and provided access to 20% sucrose. To assess the impact of biofuel crops on oviposition site selection, three oviposition cups lined with germinating paper and half-filled with infusions of corn, switchgrass, or Miscanthus leaf tissue were also installed in the cages. Senescent corn, Miscanthus, and switchgrass leaf tissues were collected from whole plants prior to harvesting. Corn leaves were obtained from nearby commercial farms while Miscanthus and switchgrass leaf tissues were obtained from the Energy Farm at University of Illinois Urbana-Champaign. Fifteen replicates were established for each species. The number of eggs laid in each type of infusion was counted and one-way anova was used to test for treatment differences in oviposition preference.
Survival and development time to adulthood experiment
First instar larvae of Ae. aegypti and Ae. albopictus (approximately 12 h old) were reared at eight intraspecific and interspecific density combinations (Ae. aegypti: Ae. albopictus; 10 : 0, 20 : 0, 40 : 0, 10 : 10, 20 : 20, 0 : 40, 0 : 20, and 0 : 10) in senescent leaf-infusion of one of the three biofuel crops: corn, switchgrass, and Miscanthus. Each container received 350 ml of deionized water and 1 g of senescent leaf litter. Each treatment was replicated four times. The containers were randomized and held in an environmental chamber at 28 °C, 70% relative humidity, and 14 : 10 light:dark photoperiod. Pupae were removed each day and allowed to eclose in vials. Eclosed adults were sexed, identified to species, and their date of eclosion recorded. Survival and development time to adulthood was calculated for each container and a general linear model was used to determine the effect of leaf litter and intra- and interspecific larval density on survival and development time to adulthood. Statistical analysis was not conducted for development time to adulthood in corn treatments due to the low number of survivors. Survival and development time data, respectively, were arcsine and log-transformed to meet the assumption of normality.
Physical and chemical characteristics
Four additional replicates were established for each crop and used to quantify the physical and chemical characteristics of the containers, including temperature (EXSTIK D0600, Extech Instruments Corporation, Waltham, MA, USA), pH, salinity, conductivity, and total dissolved solids (TDS; EXSTIK EC500, Extech Instruments Corporation). One-way anova was used to assess the effect of biofuel crops on the physical and chemical characteristics of the containers and significant means were separated by Tukey test.
Influence of biofuel crop on oviposition site selection
For Ae. aegypti, the mean number of eggs deposited in Miscanthus treatments was significantly lower than in corn and switchgrass treatments (F(2,42) = 108.80, 42, P < 0.001; Fig. 1). For Ae. albopictus, the mean number of eggs deposited in Miscanthus treatments was significantly lower than in corn, but not in switchgrass treatments (F(2,42) = 56.12, P < 0.001; Fig. 1).
Impact on life history traits
For both mosquito species, survival to adulthood was significantly influenced by a two-way interaction between leaf species and larval density (Ae. albopictus; F(8,45) = 32.98, P < 0.001; Ae. aegypti; F(8,45) = 18.26, P < 0.001). Overall, survival to adulthood was significantly lower in corn than in switchgrass and Miscanthus (Figs 2a and 3a). For both mosquito species, survival to adulthood in both switchgrass and Miscanthus treatments was consistently lower in high density treatments (0 : 40 and 20 : 20) compared to other treatments (Figs 2a and 3a). In addition, survival to adulthood in Miscanthus treatments was significantly lower at high intraspecific density (40 : 0 and 0 : 40) than at high interspecific density (20 : 20). For Ae. aegypti, but not Ae. albopictus, survival to adulthood in corn treatments was significantly lower in high density treatments (40 : 0 and 20 : 20) compared to other treatments. Due to lower survivorship in the corn infusion, development times were estimated only for switchgrass and Miscanthus treatments. Ae. aegypti females and both sexes of Ae. albopictus took longer to develop under high intraspecific and interspecific densities (0 : 40, 20 : 20) compared to the remaining treatments (Ae. albopictus; Females; F(4,28) = 156.99, P < 0.001, Males; F(8,45) = 55.43, P < 0.001, Fig. 2b and c; Ae. aegypti; F(8,45) = 88.82, P < 0.001 Fig. 3b). Ae. aegypti and Ae. albopictus females in Miscanthus and switchgrass, respectively, took longer to develop under high interspecific than high intraspecific density. Ae. aegypti males took longer to develop under high intraspecific density compared to the other treatments (F(8,45) = 27.89, P < 0.001, Fig. 3c).
Physical and chemical characteristics
There were significant treatment differences in water chemistry parameters, confirming that different biofuel crops will differently influence the chemistry of the aquatic environments in which mosquitoes develop (Table 1). Specifically, one-way anova revealed that corn treatments were characterized by low pH and high salinity, conductivity, TDS, and temperature compared to switchgrass and Miscanthus treatments. In addition, switchgrass treatments exhibited significantly higher salinity than Miscanthus treatments.
Table 1. Water chemistry parameters in corn, switchgrass, and Miscanthus treatments. Means followed by different letters are significantly different
169.0 ± 5.0a
115.3 ± 0.8b
129.0 ± 2.1c
6.5 ± 0.1a
7.1 ± 0.0b
7.1 ± 0.0b
338.2 ± 7.6a
230.0 ± 3.0b
259.2 ± 3.1c
Total dissolved solids (mg/L)
237.2 ± 6.5a
164.1 ± 0.6b
165.4 ± 1.7b
23.5 ± 0.0a
23.1 ± 0.1b
23.0 ± 0.1b
Oviposition site selection is an important component of mosquito biology; choosing habitats that are suitable for larval development is crucial for the survival of a female's progeny. The attractiveness of natural aquatic habitats to gravid female mosquitoes is influenced by multiple physical, chemical, and biological cues (Ritchie, 1984; Millar et al., 1992; Torres-Estrada et al., 2001; Sumba et al., 2008). The cues that mediate oviposition behavior originate from potential predators and competitors (e.g. conspecific and heterospecific larvae) (Torres-Estrada et al., 2001; Sumba et al., 2008) and from metabolic products of microbial decay (Benzon & Apperson, 1988). Here, we provide evidence that the biofuel crops can potentially influence the population dynamics of vector mosquitoes by altering their oviposition site selection. Gravid Ae. aegypti and Ae. albopictus females deposited significantly more eggs in corn infusions than in Miscanthus infusion. Previous studies have documented differential responses of gravid females to infusions of different plant species (Ponnusamy et al., 2010); however, this is the first study to assess the impact of biofuel crops on mosquito oviposition behavior. We attribute the differences in oviposition preference to changes in water chemistry. Corn treatments had lower pH values and higher temperature, conductivity, salinity, and TDS values than Miscanthus and switchgrass treatments. Given that switchgrass was equally attractive to both mosquito species, especially Ae. aegypti, yet it had pH, TDS, and temperature values similar to Miscanthus, our results suggest that salinity and conductivity (both of which were intermediate in switchgrass) were the most important factors influencing oviposition behavior.
Theory predicts that gravid females will maximize their fitness by depositing their eggs in high quality habitats (Jaenike, 1978) and for many taxa, there exists a strong relationship between oviposition preference and offspring performance (Mayhew, 1997). This hypothesis received little support from our data; both mosquito species had identical ovipositional preferences for corn and switchgrass infusions, yet corn had the lowest survivorship. In addition, Miscanthus was the least preferred oviposition substrate, but mosquitoes in these treatments had identical survivorship and development times as those in switchgrass infusion. Thus, it appears that attraction of gravid females to corn infusions might present an ‘ecological trap’, a phenomenon that occurs when the attractiveness of a habitat is disproportionately greater than its value for survival and reproduction (Dwernych & Boag, 1972). It remains unclear why corn was such a poor resource for the development of mosquito larvae compared to Miscanthus and switchgrass, because it is composed of rapidly degrading starches while Miscanthus and switchgrass are primarily composed of a slowly decomposing complex matrix of polysaccharides and lignins (Simmons et al., 2008). Mosquitoes have been shown to perform better in aquatic habitats containing rapidly decaying leaf species than those containing slowly decaying leaf species (Fish & Carpenter, 1982; Yee & Juliano, 2006). Corn production in the U.S. relies heavily on pesticides (USDA-NASS, 2007) and it is possible that the residual systemic neonicotinoid insecticide in senescent corn leaves may have contributed to the observed mortality-nearly all U.S. seedcorn is treated with a seed applied systemic neonicotinoid insecticide (Krupke et al., 2012). One of the two most commonly applied neonicotinoid seed treatments on corn, thiamethoxam, was shown to be toxic to Ae. taeniorhynchus, Anopheles quadrimaculatus, and Culex quinquefasciatus (Allan, 2011).
Based upon these results, it may appear that the transition to second-generation biofuel crops could increase mosquito production and consequently the risk of mosquito-borne disease. However, perennial second-generation biofuels may also influence mosquito-borne disease risk by other pathways. For example, second-generation biofuels are expected to improve wildlife diversity which may reduce infection prevalence by deflecting vector blood meals on to less competent reservoir hosts (e.g. Ostfeld & Keesing, 2000). Moreover, we used a model system comprised of laboratory colonies of container inhabiting species; Ae. aegypti and Ae. albopictus. Similar studies using field populations of Culex pipiens pipiens and Culex restuans, the primary vectors of WNV in the eastern United States, will provide further insights on this topic given that both species prefer to breed in eutrophic habitats.
As in previous studies, we found strong, negative, density-dependent effects of competition on survival and development of both mosquito species (Moore & Fisher, 1969; Black et al., 1989). In Miscanthus, the effects of intraspecific competition on survival of both mosquito species were stronger than those of interspecific competition in the high density treatments. Because mosquito larvae feed on microbial communities emanating from decaying organic matter (Fish & Carpenter, 1982), we speculate that Miscanthus may have yielded a greater diversity of microbial types and each species may have specialized on certain microbial classes thereby alleviating interspecific competition. The two species are known to differ in their larval foraging behaviors which may allow differential utilization of microbial resources (Yee et al., 2004). Interestingly, for Ae. aegypti, but not Ae. albopictus, the negative effects of interspecific competition on female development time were stronger than those of intraspecific competition in the high density treatments and the opposite was true for the males. Greater survival in high density interspecific treatments compared to high density intraspecific treatments may have triggered some form of stress (e.g. competition for food or interspecific interference) leading to longer female development times. However, males are less likely to be affected by these stresses than females because they develop faster, are smaller, and require fewer resources.
In summary, our findings demonstrate the potential for the anticipated expansion of second-generation biofuel crops to alter mosquito population dynamics and perhaps the risk of mosquito-borne disease. We recommend further studies to explore the pathways by which these crops are likely to influence disease risk so that any potential negative impacts on human health can be identified and mitigated.
We thank Nina Krasavin, Millon Blackshear, Allison Montogmery, Rosa Alfaro, Macy Brusich, James Ricci, and Thorsten Hansen for assistance with daily maintenance of the experiments. Aedes albopictus and Ae. aegypti eggs to start colonies for this research were generously provided by Dr. Philip Lounibos. The study was conducted in compliance with animal care protocol number 11121 at University of Illinois. This study was supported by the Used Tire Fund and Emergency Public Health Fund from the State of Illinois.