Introduction

The southern house mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), is a worldwide distributed vector of numerous pathogens, including Wuchereria bancrofti Cobbold (Kasili et al., 2009), Dirofilaria immitis Leidy (Labarthe et al., 1998) and avian malaria (Farajollahi et al., 2011). Besides, this mosquito is also one of the main species responsible for the transmission of the arboviruses West Nile (WNV) and St. Louis encephalitis (SLEV) viruses (Farajollahi et al., 2011). Because this, this species is of critical concern due to its impact on both public and veterinary health.

Since arboviruses are transmitted by the bite of infected mosquitoes between vertebrate hosts, activity of these pathogens is determined, at least in part, by the blood feeding habits of these vectors. According to its host feeding pattern range, Culex mosquitoes can be regarded as specialists or generalists. Specialist species include mosquitoes feeding primarily on mammals (Muturi et al., 2008), birds (Jansen et al., 2009), reptiles and amphibians (Janssen et al., 2015). Generalists are mosquitoes fed on the most abundant available hosts (Molaei et al., 2007). Additionally, some species could experience a seasonal shift in its feeding habits, from avian to mammal hosts (Edman & Taylor, 1968; Hancock & Camp, 2022), being of greater concern from an epidemiological perspective given that they can act as bridge vectors in the transmission of some arboviruses, such as SLEV and WNV (Kilpatrick et al., 2006).

West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) are zoonotic pathogens maintained in nature through an enzootic network involving transmission among viremic birds and ornithophagic mosquitoes (Kilpatrick et al., 2006). However, during late summer and early autumn, these viruses increase their activity in humans and other mammals, which has led to repeated epidemics in the US (Kilpatrick et al., 2006) and South America (Spinsanti et al., 2008). Likewise, several Culex species, including Cx. quinquefasciatus, have exhibited a seasonal shift in their feeding behaviour, feeding primarily on birds in spring and early summer and increasing the proportion of mammal blood meals in late summer and autumn (Thiemann et al., 2011). This seasonal shift suggests that viral activity might spill over from birds to mammals as a result of the mosquito host switch (Edman & Taylor, 1968; Kilpatrick et al., 2006).

Two main contrasting hypotheses have been proposed to explain why primarily ornithophagic mosquitoes increase their feeding on mammals later in the season. The migration hypothesis, proposed by Kilpatrick et al. (2006) for Cx. pipiens, suggests that this shift in feeding pattern is related to the abundance of the preferred avian host, the American robin (Turdus migratorius L.). This hypothesis is based on their findings that as robin densities decrease, the proportion of mammalian blood meals in Cx. pipiens populations increase. However, this hypothesis has several limitations: (i) multiple studies have shown that American robins are not always the preferred host for all Cx. pipiens populations (Patrican et al., 2007; Apperson et al., 2002); (ii) in some areas (e.g., Tennessee) with documented seasonal host shifts, American robins are present year-round (Savage et al., 2007); and (iii) this explanation applies only to mosquitoes whose primary host is the American robin, a species found only in the Northern Hemisphere, or another migratory bird. Therefore, it is not applicable to southern regions, such as Argentina, where SLEV seasonality is documented (Spinsanti et al., 2008) but American robins are absent. Because this, it seems that the hypothesis proposed by Kilpatrick et al. (2006) might be habitat dependent (Caranci, 2010). On the other hand, the defensive behaviour hypothesis proposed by Burkett-Cadena et al. (2011), suggests that host breeding cycles drive the host shift of mosquitoes. According to this hypothesis, in hosts with parental care, during reproductive seasons there is a greater investment of energy in assuring the offspring survivorship, consequently producing an increase in susceptibility of being bitten by mosquitoes as a result of a decrease in defensive behaviours. This event leads to the detection of peaks of host use during periods of reproductive investment, in summer for birds and autumn for mammals (Burkett-Cadena et al., 2011). While this hypothesis was proposed as an alternative to address the limitations of the migration hypothesis, it also has drawbacks. It is based on a limited range of host species and assumes a distinct seasonal phenology, which is not present for all mosquito hosts. Because this, this hypothesis seems to be host dependent.

Explanations for the seasonal shift in mosquito host feeding focused on vector biology are largely lacking in the literature. Numerous biological factors, such as stress, metabolic rate, and blood meal source, along with environmental variables like temperature and photoperiod, influence mosquito physiology and can affect reproductive traits such as fecundity, development rate, and survivorship. These factors can give rise to new nutritional requirements that may ultimately lead to seasonal variations in host selection by mosquitoes (Ciota et al., 2014; Costanzo et al., 2015; Gervasi et al., 2016; Yan et al., 2017, 2018). While several studies have examined the effects of these biological and environmental variables on mosquito reproduction, the complex nature of mosquito biology suggests that multiple interactions among these variables may produce novel responses that are not apparent when considered individually. The aim of this study was to assess whether there is an interaction between the source of the host blood meal and seasonality (defined by temperature and photoperiod) on three reproductive traits of Culex quinquefasciatus mosquitoes: fecundity, fertility, and hatchability. We hypothesize that the interaction between these two variables influence the reproductive outcomes, potentially leading to a seasonal shift in host selection, driven by a reproductive advantage. Given the reported seasonal changes in host use by Cx. quinquefasciatus, in autumn we expect a greater number of eggs (fecundity) and larvae (fertility) in mosquitoes after feeding on a mammal host compared to an avian host, and the opposite trend in summer.

Materials and methods

Establishment and maintenance of mosquitoes

Egg rafts of Culex quinquefasciatus were collected from a drainage ditch at Universidad Nacional de Córdoba Campus, Córdoba city, in February 2021. Each raft was individually maintained in plastic containers with one liter of distilled water. The hatched larvae were fed with 100 mg of liver powder three times per week until pupation. Pupae were then transferred to plastic emerging cages (21 cm x 12 cm) covered with a tulle-like fabric, containing distilled water but no food. Adults emerging from each raft were identified morphologically (Darsie, 1985) and molecularly (Smith & Fonseca, 2004) to ensure they corresponded to Cx. quinquefasciatus, since Cx. pipiens and its hybrids coexist sympatrically in Córdoba city (Branda et al., 2021). All adults were reared in a cardboard cage of 22.5 liters (28 cm x 36.5 cm) and provided ad libitum with a 10% sugar solution soaked in cotton pads placed on plastic cups. For long-term maintenance of the colony, 24-hour-starved mosquitoes were offered a blood meal from a restrained chicken twice a month. Four days after feeding, a plastic container with distilled water was placed inside the cage to allow engorged females to lay egg rafts. Batches of egg were collected and transferred to plastic containers (30 cm x 25 cm x 7 cm) in a proportion of three rafts per container, filled with three liters of distilled water. The hatched larvae were also fed with liver powder at the same proportion described above. Pupae were transferred to emerging cages, and adult mosquitoes were placed in the final cardboard cage. The colony has been maintained for several generations in the Insectary of the Instituto de Virología “Dr. J.M. Vanella” (InViV). Room conditions were controlled at 28 ± 1 °C, with a 12L:12D photoperiod and 70% relative humidity.

Experimental design

Blood source

For experimental trials, avian hosts (live chicks of the species Gallus gallus) and mammalian hosts (live mice of the species Mus musculus, strain C57BL/6) were used to evaluate the effect of blood meal source on fecundity, fertility, and hatchability. Chicks were generously donated by the Bartolucci poultry farm (Córdoba, Argentina), while mice were commercially obtained from the Instituto de Investigación Médica Mercedes y Martín Ferreyra (CONICET - Universidad Nacional de Córdoba). In each trial, 24-hour-starved adult female mosquitoes were provided with a blood meal from restrained chicks or mice. Vertebrate hosts were offered to mosquitoes one hour before the lights were turned off and were kept for 3 hours during 2 consecutive gonotrophic cycles.

The experimental use of animals (mosquitoes and vertebrates) was approved by the ethical committee at Facultad de Ciencias Médicas, Universidad Nacional de Córdoba (FCM-UNC) in compliance with the legislation regarding the use of animals for experimental and other scientific purposes (accession code: CE-2022-00518476-UNC-SCT#FCM).

Seasonality

To assess the effect of seasonality (photoperiod + temperature) on fecundity, fertility, and hatchability, a “typical” summer and autumn day from Córdoba city was simulated in an incubator where mosquitoes were housed throughout the entire experiment. Summer conditions were as follows: T°_min = 22°C, T°_max = 28°C, photoperiod = 14L:10D. Autumn conditions were characterized by: T°_min = 16°C, T°_max = 22°C, and photoperiod = 10L:14D. The humidity levels were maintained at 60-70% for both conditions. Data for simulated conditions were obtained from Climate Data website (https://es.climate-data.org/).

Feeding trial design

The interaction effect of blood source (chicken and mouse) and seasonality (autumn and summer) was evaluated during two consecutive gonotrophic cycles (I and II). This combination produced eight colonies that were established using egg rafts collected from the maintenance colony: mouse:autumn-I, mouse:autumn-II, chicken:autumn-I, chicken:autumn-II, mouse:summer-I, mouse:summer-II, chicken:summer-I, chicken:summer-II. This set of 8 colonies was repeated in triplicate (1-3), generating a total of 24 treatments.

The experimental colonies were maintained under controlled conditions to ensure that all adults were of the same size (Kauffman et al., 2017). Adult mosquitoes were placed in cardboard cages of 8.3 liters (21 cm x 24 cm) and were provided with ad libitum access to a 10% sugar solution, which was soaked in cotton pads placed on plastic cups.

Feeding trials were conducted at two time points: 5 days post-emergence (first cycle) and 14 days post-emergence (second cycle). Following each blood meal, female mosquitoes were anesthetized using CO₂ and classified as fully engorged (VI Sella’s stage), partially fed (I-V Sella’s stage), or unfed (I Sella’s stage), following the classification by Silva Santos et al. (2019). Fully engorged females were counted and separated into a separate cage to complete their gonotrophic cycle. Four days after feeding, a cup containing distilled water was placed inside the cage to facilitate oviposition. After each oviposition cycle, egg rafts were collected, counted, and transferred to a 12-well plate containing 4 mL of distilled water. They were then photographed to subsequently determine the number of eggs per raft. Rafts were maintained in each well until they hatched into L1 larvae, at which point they were preserved with 2 mL of 96% ethanol, and the number of L1 larvae per raft was counted.

Statistical analysis

The reproductive outputs measured for each experimental colony were fecundity, fertility, and hatchability. All analyses were performed using R Studio statistic software, v.4.2.1 (R Core Team, 2022).

Fecundity was defined as the number of eggs per raft and fertility as the number of L1 larvae hatched per raft. To evaluate the effect of blood meal source and seasonality on these two variables, a Generalized Linear Mixed Model (GLMM; lme4 package) with negative binomial error distribution and logarithmic link function was adjusted (Bates et al., 2015). Fecundity and fertility served as the response variables, while the fixed explanatory variables included blood source, seasonality, and gonotrophic cycle, all in a three-way interaction. Treatments (n = 24) were set as a random intercept.

Hatchability (= hatching rate) was defined as the ratio of the number of larvae to the number of eggs per raft. A Generalized Linear Model (GLM; MASS package) was applied using a quasipoisson error distribution and a logarithmic link function (Venables et al., 2002). The response variable was the number of larvae, while the explanatory variables included blood source, seasonality, gonotrophic cycle and replicate as interaction effects. Additionally, the logarithm of the number of eggs was incorporated as an offset in the model.

Multiple comparisons among treatments were conducted using Tukey’s honestly significant difference test (HSD Tukey), incorporating the Kramer (1956) correction for unbalanced data, also known as the Tukey-Kramer method. This approach allows setting the family-wise error rate to 0.05. These comparisons were performed using the emmeans package (Lenth, 2024).

Model validation was assessed using a simulation-based approach to generate randomized quantile residuals to test goodness of fit (Q-Q plot), homoscedasticity, and outliers and influential points (Cook’s distance and Leverage plots). These analyses were conducted with the DHARMa package (Hartig, 2022).

Additionally, the statistical power of the models was assessed using the mixedpower package (Kumle et al., 2021), specifically designed to evaluate the power of Generalized Linear Mixed Models (GLMMs). The analysis involved simulating 1,000 iterations with a sample size ranging from 30 to 150 observations per group. The power analysis aimed to achieve a power threshold of 0.8, indicating an 80% chance of detecting true effects if they exist.

Results

A total of 1,162 egg rafts were obtained for analysis, distributed across the eight treatments as follows: chicken:autumn-I: 260, chicken:autumn-II: 174, chicken:summer-I: 162, chicken:summer-II: 88, mouse:autumn-I: 146, mouse:autumn-II: 96, mouse:summer-I: 167, and mouse:summer-II: 69. The full count of egg rafts for each replicate (24 treatments), along with the means for fecundity, fertility, and hatchability, are summarized in Table 1. This sample size fulfilled the statistical power of 0.8 (Figure SM1).

Table 1.

In both models—fecundity and fertility—the interaction between blood source and seasonality was statistically significant (fecundity: LRT X² = 5.69, p < 0.05; fertility: LRT X² = 4.37, p < 0.05) (Tables 2,3; Figures 1,2A). Mosquitoes that fed on chicken blood had the highest fecundity (summer: 136.4 eggs/raft, autumn: 141.5 eggs/raft) and fertility (summer: 123.9 larvae/raft, autumn: 126 larvae/raft) in both seasons. In summer, fecundity and fertility were 7% and 11% higher, respectively, in chicken-fed mosquitoes compared to mouse-fed mosquitoes. In autumn, these differences increased to 46% for fecundity and 52% for fertility in favor of chicken-fed mosquitoes. No significant seasonal variation was observed in fecundity and fertility for chicken-fed mosquitoes, but both measures were statistically significant lower by 24% and 25%, respectively, in autumn compared to summer for mouse-fed mosquitoes.

Table 2.

Table 3.

Figure 1.

Figure 2.

In the fertility model, there was also a significant interaction between blood source and gonotrophic cycle (LRT X² = 3.98, p < 0.05), indicating that the impact of blood type on larvae production differed between the first and second cycles (Table 3, Figure 2B). Chicken-fed mosquitoes showed no difference in fertility between cycles, while those fed on mouse blood had 25% lower fertility in the second cycle compared to the first. During the second cycle, fertility was 50% higher in chicken-fed mosquitoes compared to mouse-fed mosquitoes.

Figure 3 presents the predicted mean values of fecundity and fertility for the 24 treatments, with no differences observed across the three replicates. The variance of random intercepts among treatments was approximately 0.006 for fecundity and 0.007 for fertility, accounting for 6% and 4% of the total variance in the respective models.

Figure 3.

There was no statistical difference among treatments and replicates in terms of hatchability, given that no statistical effects of blood source, seasonality or gonotrophic cycle was observed (Table 4). The mean hatchability for all treatments was 0.89, with a range between 0.84 for the mouse:autumn-II (replicate 1) colony and 0.96 for mouse:autumn-I (replicate 1) (Table 1, Figure 4).

Figure 4.

Table 4.

Discussion

Our results show there is an interaction effect between blood meal source and seasonality on reproductive outputs of Cx. quinquefasciatus. While our model hypothesized that fecundity and fertility would be higher in bird-fed mosquitoes than in mammal-fed mosquitoes during summer, and the reverse would occur in autumn, our findings revealed the opposite pattern. Regardless the season, fecundity and fertility in Culex quinquefasciatus were higher in bird-fed mosquitoes compared to those fed on mammals. This pattern aligns with those findings from previous studies (Akoh et al., 1993; Richards et al., 2012; Telang & Skinner, 2019). This trend is not unique to Cx. quinquefasciatus but has also been seen in other Culex species (Shroyer & Silvery, 1972) and Aedes mosquitoes (Harrison et al., 2021). The commonly accepted explanation for this pattern suggests that the nutritional quality of avian blood, with its nucleated and larger erythrocytes, provides greater protein and energy, thereby enhancing these reproductive outputs (Alto et al., 2014). However, our findings offer a mixed perspective by highlighting the interaction between blood meal source and seasonality that further might influence fecundity and fertility.

The significant interaction observed between blood meal source and seasonality revealed that mammal-fed mosquitoes had lower fecundity and fertility in autumn compared to summer, while bird-fed mosquitoes maintained similar reproductive outputs across both seasons. This finding diverges from the expectation of consistently higher fecundity and fertility in bird-fed mosquitoes and consistently lower fecundity and fertility in mammal-fed mosquitoes. This suggests that environmental factors such as temperature and photoperiod, which have been shown to affect mosquito fitness (Mordecai et al., 2019; Abouzied, 2017; Mogi, 1992), may interact with blood meal source in complex ways. Although the effects of temperature and photoperiod are generally coupled and follow a unimodal trend with an optimal peak at 28°C (Mordecai et al., 2019), our results indicate that the interaction between seasonality and blood source might play a critical role in determining reproductive success.

Additionally, our study found a significant interaction between blood meal source and gonotrophic cycle on fertility. For mammal-fed mosquitoes, fertility was significantly lower in the second gonotrophic cycle compared to the first, a pattern commonly reported in other studies where the first and second cycles are generally the most productive (Awahmukalah & Brooks, 1985; Christiansen-Jucht et al., 2015). This declining fertility pattern has been documented in Cx. quinquefasciatus fed on both birds and mammals (Richards et al., 2012; Telang & Skinner, 2019). However, our results showed that this decline in fertility was not present in bird-fed mosquitoes, indicating a potential role of blood meal source in moderating reproductive output across gonotrophic cycles.

We also investigated whether hatchability could account for the observed variations in fertility among different treatments. Our results indicate no difference in hatchability among treatments.

While our findings corroborate an interaction effect between blood meal source and seasonality, they are not without caveats, as same as other studies. First, our research was conducted under controlled conditions in a laboratory incubator, which implies that our results may differ from those of field studies. Some of the main limitations of this approach include the use of artificial, constant light and temperature rather than a fluctuating environment, and the use of constant humidity, which could affect mosquito performance. However, this is the first study of its kind, and future investigations should aim for more realistic setups. A second limitation, commonly encountered in ecological and behavioral studies, is determining an appropriate sample size for each treatment, as this can impact the ability to detect desired effects or interactions (Taborsky, 2010). To address this issue, we conducted an a priori power analysis to determine the minimum required sample size and repeated the assay multiple times (three replicates). Furthermore, the presence of interaction effects among our explanatory variables suggests that our chosen sample size may have been adequate. Lastly, another aspect of this research that warrants further discussion is the underlying hypothesis that Culex mosquitoes change their feeding habits from birds to mammals. Given that most species are ornithophagic, why is this shift not directed from one bird host to another? In fact, both patterns—bird-to-mammal (Burkett-Cadena et al., 2011) and bird-to-bird (Kent et al., 2009) shifts—are mentioned in the literature. Future studies are aimed to clarify the complex feeding behaviour of Culex mosquitoes regarding seasonal switching, which may depend on several factors, such as habitat, as evidenced in U.S. Culex populations (Caranci, 2010).

In summary, our study investigates the critical feature of host shifting patterns in Culex mosquitoes, which holds significant importance due to their role as bridge vectors between bird and human hosts. Although our study detects a significant interaction between blood meal source and seasonality, we observed the opposite expected trend, suggesting a complex interplay of factors affecting mosquito reproduction and its relationship with host use. We could consider three possible explanations for our results. Firstly, it is possible that additional factors, such as genetic mechanisms or variations in midgut microbiota composition play a role in influencing host use patterns. Secondly, host switching might be explained by factors related to vector biology other than fitness. Lastly, it remains a possibility that Argentinian Cx. quinquefasciatus populations do not exhibit the same host-switching behaviour observed in US populations, and the observed interaction effects may be linked to different mosquito behaviours. However, Spinsanti et al. (2008) found a seasonal variation in WNV human cases, and Beranek (2019) also observed that in autumn, the proportion of blood meals was higher than in summer for Culex quinquefasciatus. Therefore, more in-field studies are needed to assess seasonal host variation in Culex mosquitoes from Argentina.

Further investigations are warranted to corroborate our findings and gain a deeper understanding of the interaction between mosquito feeding pattern and seasonality. Last, these investigations will provide valuable insights into mosquito-borne disease transmission and forecasting its emergence.

Data availability

All data generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgements

We would like to thank Dr. Agustín Quaglia for providing valuable help in statistical analysis. In addition, we greatly appreciate very helpful comments on the manuscript by anonymous reviewers. This study was supported by grant PICT 2018-1172 (FONCYT) and Consolidar Secyt-UNC (2018-2023).

Competing interests

The authors declare no competing interests.

Author contributions

Kevin Alen Rucci, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization; Gabriel Barco, Investigation, Data Curation, Writing – Review & Editing; Andrea Onorato, Investigation, Data Curation, Writing – Review & Editing; Mauricio Beranek, Investigation, Data Curation, Writing – Review & Editing; Mariana Pueta, Conceptualization, Methodology, Validation, Writing – Review & Editing, Visualization, Supervision; Adrián Diaz, , Conceptualization, Methodology, Validation, Writing – Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.