Widespread evidence for interspecific mating ...

MEEGID-02445; No of Pages 6 Infection, Genetics and Evolution xxx (2015) xxx–xxx

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Widespread evidence for interspecific mating between Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in nature I.E. Bargielowski a,⁎, L.P. Lounibos a, D. Shin a, C.T. Smartt a, M.C. Carrasquilla a, A. Henry b,c, J.C. Navarro d,1, C. Paupy e,2, J.A. Dennett f a

University of Florida, Florida Medical Entomology Laboratory, 200 9th Street, Vero Beach, FL 32962, USA Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School Singapore, 8 College Road, Singapore 169857, Singapore Department of Microbiology, University of Hawai'i at Manoa, Honolulu, 2500 Campus Rd, Honolulu, HI 96822, USA d Universidad Central de Venezuela, Instituto de Zoología y Ecología Tropical, Apdo. 47058, Caracas, Venezuela e Centre International de Recherches Médicales de Franceville (CIRMF), BP 769, Franceville, Gabon f Harris County Public Health & Environmental Services, Mosquito Control Division, 3330 Old Spanish Trail, Building ‘D’, Houston, TX 77021, USA b c

a r t i c l e

i n f o

Article history: Received 18 May 2015 Received in revised form 9 August 2015 Accepted 11 August 2015 Available online xxxx Keywords: Aedes aegypti Aedes albopictus Reproductive interference Satyrization Reproductive fitness Displacement Competitive exclusion

a b s t r a c t Aedes aegypti and Aedes albopictus, two important vectors of the dengue and chikungunya viruses to humans, often come in contact in their invasive ranges. In these circumstances, a number of factors are thought to influence their population dynamics, including resource competition among the larval stages, prevailing environmental conditions and reproductive interference in the form of satyrization. As the distribution and abundance of Ae. aegypti and Ae. albopictus have profound epidemiological implications, understanding the competitive interactions that influence these patterns in nature is important. While evidence for resource competition and environmental factors had been gathered from the field, the evidence for reproductive interference, though strongly inferred through laboratory trials, remained sparse (one small-scale field trial). In this paper we demonstrate that low rates (1.12–3.73%) of interspecific mating occur in nature among populations of these species that have co-existed sympatrically from 3 to 150 yrs. Finally this report contributes a new species-specific primer set for identifying the paternity of sperm extracted from field collected specimens. © 2015 Published by Elsevier B.V.

1. Introduction Reproductive interference, indiscriminate interspecific sexual interaction that reduces reproductive success, has been documented for many taxa, including plant and animal species (reviewed in Gröning and Hochkirch, 2008). Biological invasions, in particular, often lead to reproductive interference (Gröning and Hochkirch, 2008), which can result in significant consequences for the population dynamics of the species involved (Crowder et al., 2011). The interactions between Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) are a good example of this phenomenon. The spread of Ae. aegypti from Africa occurred mostly during the 15–17th centuries, leading to the establishment of this species throughout much of the Americas (Lounibos, 2002; Tabachnick, 1991), ⁎ Corresponding author. E-mail addresses: irka@ufl.edu (I.E. Bargielowski), lounibos@ufl.edu (L.P. Lounibos), dshin@ufl.edu (D. Shin), ctsmart@ufl.edu (C.T. Smartt), mccarrasquilla@ufl.edu (M.C. Carrasquilla), [email protected] (A. Henry), [email protected] (J.C. Navarro), [email protected] (C. Paupy), [email protected] (J.A. Dennett). 1 Present address: Universidad Central del Ecuador, Centro Internacional de Zoonosis, Jerónimo Leiton y Gatto Sobral. Quito, Ecuador. 2 Present address: MIVEGEC Unit, UMR 224-5290 CNRS-IRD-UM, Centre IRD de Montpellier, BP 64501, 34394 Montpellier, France.

followed by spreading into Asia in the second half of the nineteenth century (Smith, 1956; Tabachnick, 1991), while the cosmopolitan diaspora of Ae. albopictus from Asia by transcontinental shipping is largely a 20–21st century phenomenon (Benedict et al., 2007). Both species belong to the subgenus Stegomyia and their similar life histories and mating habits may contribute to interspecific mating on account of incomplete species recognition. Interspecific matings between these species do not produce viable offspring (Leahy and Craig, 1967; Lee et al., 2009) and leave females of Ae. aegypti, but not Ae. albopictus, refractory to further mating (Tripet et al., 2011). Where invasion processes have led to sympatry of these species, competitive displacement of Ae. aegypti by Ae. albopictus has been documented (e.g. Kaplan et al., 2010; Nasci et al., 1989; O'Meara et al., 1995), leading to patchy distributions (e.g. Braks et al., 2003; Higa et al., 2010; Rey et al., 2006). Larval resource competition became the preferred explanation for displacement of Ae. aegypti by Ae. albopictus (Juliano and Lounibos, 2005), however the rapidity of competitive replacements strongly suggested that larval competition alone could not be the sole mechanism (Kaplan et al., 2010; Tripet et al., 2011). Recent studies (Bargielowski et al., 2013; Bargielowski and Lounibos, 2014; Tripet et al., 2011) have shown that interspecific reproductive interference, in the form of satyrization, asymmetrically favoring Ae.

http://dx.doi.org/10.1016/j.meegid.2015.08.016 1567-1348/© 2015 Published by Elsevier B.V.

Please cite this article as: Bargielowski, I.E., et al., Widespread evidence for interspecific mating between Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in nature, Infection, Genetics and Evolution (2015), http://dx.doi.org/10.1016/j.meegid.2015.08.016

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I.E. Bargielowski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx

albopictus over Ae. aegypti is likely the driving mechanism behind these rapid displacements. Models of the outcomes of reproductive interference have shown it to be a stronger force for competitive exclusion than resource competition (Feng et al., 1997; Kishi and Nakazawa, 2013; Kuno, 1992; Ribeiro and Spielman, 1986; Yoshimura and Clark, 1994), and there are growing empirical data from arthropod and plant systems to back these model predictions (Bargielowski et al., 2013; Bargielowski and Lounibos, 2014; Crowder et al., 2010, 2011; Kishi et al., 2009; Takafuji et al., 1997; Takakura et al., 2009; Takakura and Fuji, 2010; Runquist and Stanton, 2012). Furthermore, Kishi and Nakazawa (2013) demonstrated synergy between resource competition and reproductive interference, leading to greater negative effects than the sum of the two factors alone. This finding may contribute towards explaining the rapidity with which some recently established invasive species have been able to displace resident species. The rapid reduction in range and abundance of Ae. aegypti (Hobbs et al., 1991; O'Meara et al., 1995) following the invasion and spread of Ae. albopictus throughout most of the southeastern USA in the 1980s (Craig, 1993; Hawley et al., 1987), where population displacements of Ae. aegypti occurred within 1–3 years (Hawley et al., 1987; Nasci et al., 1989; Tripet et al., 2011) and the more recent displacement of Ae. aegypti by Ae. albopictus in Bermuda with comparable rapidity (Kaplan et al., 2010) might be examples of this synergistic interaction affecting population dynamics. However, not all encounters lead to the exclusion of Ae. aegypti, and Ae. aegypti persists in southern peninsular Florida (Juliano et al., 2002, 2004; O'Meara et al., 1995) and in other southern cities including Houston, Savannah and New Orleans (Juliano and Lounibos, 2005). In these areas, the two species can be found in sympatry (Juliano et al., 2004). In some cases Ae. aegypti predominates and Ae. albopictus has failed to become established (e.g. in urban Miami (Lounibos et al., 2010; O'Meara et al., 1995) and in the Florida Keys). Patterns in the southern US, therefore, reflect the varied distribution patterns of these two species where they co-occur around the world (Bargielowski et al., 2013; Benedict et al., 2007; Craig, 1993; Higa et al., 2010; Raharimalala et al., 2012) and it is likely that a number of factors influence outcomes where these species co-exist. While field-based experiments have weighed the contributions of larval competition to the outcomes of this species interaction (reviewed in Juliano and Lounibos, 2005), the evidence for reproductive interference so far has mainly been gathered from laboratory-based studies (e.g. Bargielowski et al., 2013; Bargielowski and Lounibos, 2014; Harper and Paulson, 1994; Lee et al., 2009; Nasci et al., 1989). These trials often take the form of non-choice experiments (in which no conspecific mates are present), are carried out in cages at artificial densities of unclear relevance to natural densities, and are typically free from any natural environmental fluctuations. Extrapolating interspecific mating rates in cages to wild populations is therefore problematic. One small-scale study conducted in Florida by Tripet et al. (2011) is, to our knowledge, the only fully field-based assessment of interspecific mating between Ae. aegypti and Ae. albopictus. One hundred and seventy Ae. aegypti and 134 Ae. albopictus females, collected from two salvage yards where these species co-occur in sympatry, were screened for cross-insemination, which was found in three Ae. aegypti and two Ae. albopictus females. In this paper we document interspecific mating between these two species on a larger scale. We selected four geographically diverse locations in which Ae. aegypti and Ae. albopictus have been in sympatry for various durations (Caracas, Venezuela (approx. 3 years); Franceville, Gabon (approx. 5 years); Houston, TX, USA (approx. 25 years); Singapore (approx. 150 years)). We analyzed females of both species collected between 2011 and 2013 for paternity of extracted sperm using techniques similar to Tripet et al. (2011) to assess whether interspecific mating occurs at these sites and whether rates of reproductive interference change over time since sympatry. We hypothesized that interspecific mating would be detected at all sites, as recent models

have shown the possibility for cross mating to persist, even in the face of apparent costs (Takakura et al., 2015), however, we expected to see a decline in the frequency of satyrization with increasing time spent in sympatry, as the cost (loss of future reproductive potential) of engaging in interspecific mating is high for Ae. aegypti females. 2. Materials and methods 2.1. Field collections Collections of Ae. aegypti and Ae. albopictus were carried out using multiple trapping techniques, at sites of sympatry (Table 1). Trapped mosquitoes were sexed, identified to species and females were stored in ethanol for shipment, or ground transportation (from Houston, TX), to the Florida Medical Entomology Laboratory for species-specific polymerase chain reaction (PCR) diagnostics. 2.1.1. Caracas, Venezuela Mosquitoes (212 Ae. aegypti and 118 Ae. albopictus) were collected in 2012 with human bait (approved by the Ethics Committee, Faculty of Science, Central University of Venezuela, Project: Widespread evidence for interspecific mating between Ae. aegypti and Ae. albopictus in nature) in Caracas where Ae. albopictus had recently (2009) become established (Navarro et al., 2009, 2013). We estimate the duration of sympatry with Ae. aegypti at Caracas cemetery, where collections were carried out, to be approximately three years at the time of trapping. Both species are invasive in Venezuela, however, Ae. albopictus is the more recently established species. 2.1.2. Franceville, Gabon Collections (111 Ae. aegypti and 222 Ae. albopictus) were made with BG-Sentinel® traps or human bait (approved by the Ministère de la Recherche Scientifique et du Développement Technologique du Gabon: N° AR0006/12/MENERSI/CENAREST/CG/CST/CSAR) in three Franceville neighborhoods (Sable, Akou, Potos) in May 2012. In Franceville the two species have been in sympatry for approximately five years (C. Paupy, personal communication, Coffinet et al., 2007; Krueger and Hagen, 2007). Ae. aegypti is native to Gabon, while Ae. albopictus is invasive. 2.1.3. Houston, TX, USA Ae. albopictus was first recorded in Houston Texas in 1985 (Sprenger and Wuithiranyaggol, 1986) and we therefore estimate that the two species had been living in sympatry for over 25 years at the time of our 2012 collections. Mosquitoes (305 Ae. aegypti and 110 Ae. albopictus) were collected with BG-Sentinel® traps baited with ca. 0.90 kg dry ice within 1.89 L beverage coolers from 5 individual sites within three adjoining Houston neighborhoods. Both species are invasive in the US, however, Ae. albopictus is the more recent invader. 2.1.4. Singapore In contrast to our other collections, at this site, Ae. albopictus is the native species and Ae. aegypti the invasive, the latter believed to have established approximately 150 years ago (Smith, 1956). Collections (227 Ae. aegypti and 275 Ae. albopictus) were made in 2011 in Ang Mo Kio GRC (Group Representation Constituency), central Singapore, in five subdivisions: Teck Ghee, Kebun Baru, Cheng San, Jalan Kayu, and Yio Chu Kang (National University of Singapore, IRB10-060). In areas where BG-Sentinel® traps were likely to be stolen, adult sticky traps (MosquiTRAPsTM) were substituted. Only the abdomens of collected females were preserved and shipped from Singapore to Florida, as the heads and thoraces were retained for use in other research. 2.2. Analysis of sperm bundles The storage of females in ethanol caused the proteinaceous fluid in the spermathecae to coagulate, forming a “sperm bundle” (Tripet



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Table 1 Proportion of Ae. aegypti and Ae. albopictus trapped at four locations. The proportions of mated females are calculated using only females that were in sufficiently good condition for presence or absence of sperm to be recorded accurately, all damaged, unmated or degraded specimens were excluded from analysis. Location

Caracas, Venezuela Franceville, Gabon Houston, TX, USA Singapore, Singapore a

Years of sympatrya

3 5 25 150

Relative abundance in traps

Proportion of mated females

Ae. aegypti

Ae. albopictus

Ae. aegypti

Ae. albopictus

0.64 0.33 0.73 0.45

0.36 0.67 0.27 0.55

0.92 0.76 0.92 0.95

0.84 0.75 0.67 0.91

Approximation.

et al., 2011). Females were stored in ethanol until 12–24 h before dissection when they were transferred to a 12-well plate (MultiwellTM, Falcon®) containing water for rehydration, which enabled easier dissection. Each female was dissected with micro-pins in a drop of water under a binocular microscope. Micro-pins were sterilized between steps to prevent cross contamination. The three spermathecae were removed, cleaned of excess tissue, and placed in a fresh drop of water. Using the micro-pins the spermathecal shells were ruptured and the sperm bundle/s moved to a third drop of water to remove any contamination (of maternal DNA). The sperm bundle/s were collected on the tip of a micro-pin and transferred to 0.5 mL Eppendorf tubes containing 100 μL of lysis buffer from a Wizard® SV Genomic DNA Purification System (Promega) extraction kit and processed following kit instructions with minor modifications. Though the analysis of individual spermathecae may offer insights into spermathecal use during interspecific mating (see Tripet et al., 2011), owing to logistical constraints, when more than one spermatheca contained sperm, we pooled sperm bundles by female. Following DNA extraction, samples were stored at −20 °C until PCR analysis.

series of 20 trials for each cross combination (female Ae. aegypti × male Ae. albopictus and vice versa). Each female was dissected and analyzed as described above. In two cases (one Ae. aegypti female and one Ae. albopictus female) both paternal and maternal DNA were amplified. Therefore, while our methods are relatively (95%) accurate, and we are certain that these cases, too, are definitive cases of interspecific mating, we cannot rule out that suspected multiple inseminations may occasionally represent an artifact of maternal DNA contamination.

2.3. Species-specific PCR diagnostics

3.1. Spermathecal dissections

In general, our diagnostic methods followed those of Tripet et al. (2011), but we developed new species-specific primer sets that yielded more reliable results in our laboratory. The species-specific primers were designed based on a ribosomal gene intergenic spacer region from Ae. aegypti and Ae. albopictus (GENBANK accession numbers: AF004986 and M65063, respectively) with Primer 3 software. The primer set for Ae. aegypti (forward: 5′-GTGCGTGGACTTCTCTCT TT-3′, reverse: 5′ TTTCTCTTTCCCGACACTACA-3′) and for Ae. albopictus (forward: 5′-CACCCGTGTATGTGCGATATTA-3′, reverse: 5′-TTGGTCGT TCGGTGGTAAAG-3′) amplify a 233 base pair and 812 base pair fragment, respectively. All primers had high priming efficiency resulting in sharp specific bands easily scored on an agarose gel. The PCR diagnostic methods were tested against DNA extracts of whole bodies, sperm bundles and testes of each species. No cross-amplification was observed in tests. In cases where interspecific mating was suspected based on the analysis of sperm bundles, the same PCR diagnostics were applied to the bodies of the females from which the spermathecae had been dissected to confirm accurate identification of field-collected specimens.

Spermathecae were dissected from a total of 855 Ae. aegypti and 725 Ae. albopictus females from 4 sites, however only 572 Ae. aegypti and 299 Ae. albopictus specimens contained sperm or were in satisfactory condition to extract usable sperm bundles (Table 2). Ae. albopictus females appeared more sensitive to degradation with 58.76% of females unsuitable for analysis, versus 33.10% for Ae. aegypti females.

2.4. Dissection quality To avoid contamination, females were dissected in batches; all the Ae. aegypti females of a region, followed by all the Ae. albopictus females. Any cross contamination would therefore have to be transferred from one sperm bundle to the next. We did not detect interspecific insemination in any two consecutively dissected females and, therefore, can rule out any incidence of contamination between females. We are confident that all recorded interspecific matings are genuine. Furthermore, to test the assumption that our dissection methods were sufficiently stringent to avoid contamination with maternal DNA, we force-copulated female mosquitoes with heterospecific males (methods modified from McDaniel and Horsfall, 1957) and performed a

2.5. Statistical analysis Statistical analyses were performed using JMP (version 11) http:// www.jmp.com. Average interspecific insemination rates (arcsine transformed proportions) of Ae. aegypti and Ae. albopictus females were analyzed by ANOVA, while interspecific mating frequencies within species at sites where satyrization was observed, were compared using contingency table analyses. 3. Results

3.2. Sperm extraction The proportion of females that contained sperm whose DNA we could successfully extract, amplify, and identify to species ranged from 30% (Ae. aegypti, Singapore) to 85% (Ae. aegypti, Caracas). The relatively low numbers of usable specimens in the Singapore collections may be partially attributable to the use of sticky traps (N 80% mosquitoes received (n = 403)) at this location. Only 29% of females collected from sticky traps were in satisfactory condition for dissection and sperm extraction, versus 47% of females from BG-Sentinel® traps at this location. For future experiments we recommend using trapping methods that preserve the females as well as possible (i.e. traps that minimize desiccation) in addition to rapid preservation of samples (transfer from traps to alcohol). Overall, if sperm bundles were extracted, our average success rate of identifying paternity was 97%. Ae. albopictus dominated in collections from Franceville and Singapore, while Ae. aegypti was relatively more abundant in Caracas and Houston neighborhoods that were sampled (Table 2). 3.3. Interspecific mating rates Though on average Ae. aegypti (2.17% ± 0.57 SE) and Ae. albopictus (1.22% ± 0.10 SE) females showed similar (F = 1.19, df = 1, p = 0.34) interspecific mating rates at sites where satyrization was observed, Ae.


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Table 2 Results of genetic analysis of sperm content dissected from field-collected female mosquitoes from four locations. Location

Species (♀)

Females dissected

Females analyzed

Failed DNA extraction

Conspecific mating only

Heterospecific mating only

Mated both

% interspecific mating

Caracas, Venezuela

Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus Ae. aegypti Ae. albopictus

212 118 111 222 305 110 227 275

188 88 69 77 244 40 71 94

8 1 6 1 3 1 2 5

177 87 62 75 232 39 68 88

1 0 1 1 5 0 1 1

2 0 0 0 4 0 0 0

1.67 0 1.59 1.32 3.73 0 1.45 1.12

Franceville, Gabon Houston, TX, USA Singapore, Singapore

aegypti females engaged in interspecific mating at all four sites, while Ae. albopictus females were only documented to engage in interspecific mating at two sites (Franceville and Singapore) (Table 2). Contingency analysis showed that within species, the interspecific mating rates did not vary significantly among sites where interspecific mating occurred (Ae. aegypti: chi-square = 2.526, df = 3, p = 0.47, Ae. albopictus: chi-square = 0.013, df = 1, p = 0.91). 4. Discussion 4.1. Occurrence of interspecific mating Ae. aegypti and Ae. albopictus are considered the most invasive mosquitoes in history (Juliano and Lounibos, 2005) and, owing to their wide dispersal, often come in contact in their invasive ranges. They are the most important vectors of dengue and chikungunya viruses to humans (Kyle and Harris, 2008; Paupy et al., 2010), therefore understanding and predicting the competitive interactions that influence their distribution patterns in nature are important. Recent studies (Bargielowski et al., 2013; Bargielowski and Lounibos, 2014; Tripet et al., 2011) have suggested that interspecific reproductive interference may be a key factor in their population dynamics in sympatry, yet evidence from the field was still sparse. In this paper we provide evidence that reproductive interference may occur wherever these two species come in contact in nature. Including the two sites in Floridas sampled by Tripet et al. (2011), similar rates of interspecific mating (between 1.12 and 3.73%) have now been detected at six locations in which the two species have remained in sympatry for a varying number of years. While interspecific insemination rates of Ae. aegypti and Ae. albopictus were similar at sites where interspecific matings were recorded, Ae. aegypti mated interspecifically at all sites, while Ae. albopictus females were only recorded doing so at half the sites. Laboratory studies have demonstrated a bias in the asymmetric nature of cross-matings in favor of Ae. albopictus, (Bargielowski et al., 2013; Nasci et al., 1989) which may be weakly supported by these findings. Recent findings by Soghigian et al. (2014) suggest that asymmetric reproductive interference in this species pair may extend to deterring feeding by Ae. aegypti females. Aggressive and non-selective behavior of Ae. albopictus males leading to the harassment of Ae. aegypti females during feeding, either for sugar or blood, can reduce Ae. aegypti feeding success and, therefore, may adversely affect fecundity (Soghigian et al., 2014). 4.2. Temporal changes in interspecific mating rates As laboratory trials have shown the development of resistance to satyrization in Ae. aegypti with continued exposure to Ae. albopictus, along with an associated reduction in interspecific insemination rates (Bargielowski and Lounibos, 2014), it is likely that when these species first come in contact, interspecific mating is more frequent than the rates observed in this study. Additionally, cross-mating based on detection of interspecific sperm in spermathecae underestimates the extent

of satyrization of Ae. aegypti by Ae. albopictus, which can occur without spermathecal filling (Carrasquilla and Lounibos, in press). The strong costs associated with satyrization—loss of future reproductive potential for females and a loss of time, energy and limited sperm reserves for males, promote the avoidance of interspecific mating in both sexes (Bargielowski and Lounibos, 2014; Bargielowski et al., 2015). The rapid evolution of reproductive character displacement suggests that the strong selection pressure of satyrization can drive rapid evolutionary change in this system. This assumption is backed by comparisons of allopatric strains of Ae. aegypti and strains that were colonized from sites of recent sympatry with Ae. albopictus (Bargielowski et al., 2013), as well as the results from artificial selection experiments (Bargielowski and Lounibos, 2014), in which significant levels of satyrizationresistance were observed within 3–6 generations. Experiments in other systems, including numerous examples from the field (reviewed in Endler, 1986; Thompson, 1998) are informative examples of the speed at which evolution can reshape community structure. We therefore suspect that in areas where these two species occur in sympatry, the shift from ‘naïve’ populations, susceptible to interspecific mating errors, to populations that have reduced cross-mating to a low frequency, occurs quickly, likely within a few generations of exposure. Consequently, we were unable to detect any differences in the interspecific insemination rates from the locations sampled in this study, where sexual selection has presumably already reduced rates of satyrization. An experimental approach to following the evolution of satyrization-resistance might be the observation of changes in interspecific mating frequency of populations held in large, walk-in cages capable of recreating a semi-natural environment. 4.3. Reproductive interference, population dynamics and other environmental factors Though population reductions or displacements of Ae. aegypti by Ae. albopictus, likely driven by asymmetric effects of satyrization, are seemingly a common result of encounters between these species (Kaplan et al., 2010; Lounibos, 2002; Nasci et al., 1989; O'Meara et al., 1995), several mechanisms may allow for the persistence or recovery of Ae. aegypti populations. One such factor is the evolution of satyrization-resistance discussed above. However, this form of reproductive character displacement observed in Ae. aegypti carries with it a cost in terms of reproductive success. Although it remains uncertain which cues or behaviors change during this process, our recent work (Bargielowski and Lounibos, 2014) has detected that satyrization-resistant females are choosier during mate selection. This trait may, therefore, only persist in the presence of satyrization pressure, but the reduced interspecific mating rates of satyrization-resistant Ae. aegypti may allow for the recovery of competitively reduced populations of this species, regardless of associated costs. In addition to the evolution of satyrization resistance, resource competition and local environments may influence the distribution patterns of these two species. Resource competition among the larval stages of Ae. aegypti and Ae. albopictus in foodlimited container habitats has been shown to favor Ae. albopictus



(Barrera, 1996; Daugherty et al., 2000), while substrate-rich habitats negate this advantage and in some cases provide an advantage to Ae. aegypti (Barrera, 1996; Daugherty et al., 2000 and references therein, Juliano, 2009). Furthermore, larval competition has been shown to asymmetrically impact adult life history traits under low-humidity conditions, leading to reduced longevity of Ae. aegypti, but not Ae. albopictus (Reiskind and Lounibos, 2009). Likewise, habitat preferences (Rey et al., 2006) and climatic factors may have a role to play (Alto and Juliano, 2001; Juliano et al., 2002). Studies suggest that dryer environments may favor Ae. aegypti due to greater mortality of Ae. albopictus eggs (Juliano et al., 2002; Lounibos et al., 2010). Asymmetry in the impact of reproductive interference and resource competition on affected species pairs is not unusual (Crowder et al., 2011; Gröning and Hochkirch, 2008), and has been shown by Kishi and Nakazawa (2013) to allow for scenarios where trade-offs between asymmetries in reproductive interference and resource competition may allow for coexistence of interacting species. Thus, an inferior resource competitor may be able to persist when its disadvantage is counterbalanced by superior reproductive interference and vice versa (Kishi and Nakazawa, 2013). Therefore, understanding the interplay between reproductive interference and other factors determining the competitive outcome of Ae. aegypti and Ae. albopictus interactions may be valuable in predicting the distribution patterns of these two species. 4.4. Multiple inseminations Out of the 16 cases of interspecific mating that we detected, the spermathecal content of six females (all Ae. aegypti), tested positive for the presence of both conspecific and heterospecific DNA, suggesting multiple inseminations. Though Ae. aegypti females are considered to generally mate only once, instances of multiple insemination have been documented, particularly in cases where (a) matings followed in quick succession, before the female becomes refractory due to the effects of male accessory gland proteins; (b) in older females, where the effects of accessory gland proteins have worn off, and (c) in females mated to depleted males, that do not pass on a full complement of sperm and accessory gland substances during mating (Bargielowski, 2011; Craig, 1967; Spielman et al., 1967; Young and Downe, 1982). It is plausible that interspecific mating attempts may be more likely to fall into one of these categories, as sperm transfer between distantly related species may be suboptimal or females may abort copulation with heterospecifics and subsequently seek mating opportunities with conspecific males. Tripet et al. (2011) also documented cases of multiple insemination between Ae. aegypti and Ae. albopictus at auto salvage yards in Florida with distinct patterns of spermathecal use in cross-matings. In Ae. aegypti, heterospecific sperm and conspecific sperm were clearly separated between spermathecae; with conspecific sperm stored in the primary, large spermatheca and heterospecific sperm stored separately in one of the two lateral spermathecae. In contrast, in Ae. albopictus females, both conspecific and heterospecific DNA was amplified from the contents of one of the lateral spermathecae. 5. Conclusions In summary, the distribution and abundance of Ae. aegypti and Ae. albopictus, which have profound epidemiological implications, are often governed by competitive interactions that include interspecific mating, documented herein as widespread. We further contribute a new species-specific primer set for determining the paternity of sperm samples, and offer evidence that reproductive interference in the wild contributes to the observed distribution patterns of this species pair. We suggest that satyrization-resistance develops rapidly in sympatric populations of Ae. aegypti and Ae. albopictus, resulting in low levels of reproductive interference.

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Acknowledgments This research was supported by National Institutes of Health R21 grant AI095780 (to LPL) and in part by funding provided by the University of Hawaii's National Science Foundation Integrative Training in Ecology, Conservation and Pathogen Biology program (NSF IGERT Award #0549514). We thank Chris L. Fredregill, Greg C. Motl, and Christine Roberts for their assistance during Houston field collections, Davy Jiolle, Nil Rahola, Amadou Sekou Traoré and Patrick Yangari for sampling in Gabon, and E. Villarroel in Caracas. References Alto, B.W., Juliano, S.A., 2001. Precipitation and temperature effects on populations of Aedes albopictus (Diptera: Culicidae): implications for range expansion. J. Med. Entomol. 38, 646–656. Bargielowski, I.E., 2011. Sexual ecology of transgenic mosquitoes, Stegomyia (Aedes) aegypti (Thesis Ph.D.) Department of Biology, Imperial College London, pp. 66–76. Bargielowski, I.E., Lounibos, L.P., 2014. 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