Skip to main content
NCBI home page
Journal of Insect Science logoLink to Journal of Insect Science
. 2026 Jul 30;26(4):ieag079. doi: 10.1093/jisesa/ieag079

Thermal sensitivity in a symbiont: effect of elevated cyclic temperature exposure on Wolbachia density in Culex quinquefasciatus (Diptera: Culicidae) embryos

Jovany Barajas 1, Shaoming Huang 2, Fabian Gonzalez 3, Andrea Joyce 4,✉
Editor: Teresa Bonacci
PMCID: PMC13424437  PMID: 42533853

Abstract

Temperature influences insect reproduction and the density of the maternally inherited bacterium Wolbachia, which in turn manipulates host reproduction and alters arbovirus transmission. Wolbachia is used in mosquito control strategies, and thermal conditions may shape Wolbachia densities. Species-specific thermal tolerances have been documented in mosquitoes but little is known of thermal impacts on embryos. This study investigated Wolbachia density under elevated cyclical temperatures in Culex quinquefasciatus Say embryos, specifically to determine whether high temperatures reduced Wolbachia density. Experiments used cyclic thermal regimes that simulated high summer temperatures similar to those observed where Cx. quinquefasciatus occurs. The relationship between temperature and Wolbachia density was determined through Fluorescent in-situ hybridization (FISH) and relative quantification based on qPCR. The embryo developmental time and Wolbachia density were reduced at the high cyclic temperature (25 to 40 °C) when compared to the control (25 °C). The time for the embryonic maturation at the high cyclic temperature (25 to 40 °C) was reduced by 8 h compared to development at 25 °C. Integrated fluorescence density of FISH-labeled Cx. quinquefasciatus embryos was significantly lower under the elevated cyclic 25 to 40 °C temperature than at control 25 °C. Relative Wolbachia density by qPCR and ΔΔCt of the wsp gene was also lower under the cyclic elevated temperature. Elevated temperatures which reduce Wolbachia loads may disrupt cytoplasmic incompatibility or antiviral protection. The results herein suggest that environmental thermal regimes could disrupt Wolbachia density in mosquito embryos. This could have implications for Wolbachia‑based disease control strategies and merits further investigation.

Keywords: Culex pipiens complex, heatwaves, vector control, symbionts, climate change

Introduction

Culex quinquefasciatus Say (1823) (Diptera: Culicidae), also referred to as the common house mosquito, is one of the most widely distributed mosquito species globally, and is found in temperate and tropical regions spanning America, Europe, Asia, and Africa. Rising global temperatures may increase its geographic distribution and the area vulnerable to its vector-borne diseases (Salim et al. 2024, Saarman et al. 2025). Its adaptability to diverse environments, from urban areas to natural wetlands, enables it to exploit a wide range of breeding sites and to thrive in areas where summer ambient air temperatures routinely exceed 35 to 40 °C, including the Central Valley of California, the Southern United States, parts of the Mediterranean and much of South Asia and sub-Saharan Africa (Rasgon and Scott 2003, Hernandez et al. 2019, Iwamura et al. 2020, Tokash-Peters et al. 2022). These hot, often densely populated regions are the same settings in which Cx. quinquefasciatus transmits West Nile virus (WNV), St. Louis encephalitis and Japanese encephalitis virus (Kramer et al. 2008, Bartholomay et al. 2010). The mosquito is also a competent vector of the filarial nematodes that cause lymphatic filariasis in tropical Africa, South Asia, and the Western Pacific (Ledesma and Harrington 2011, Huang et al. 2013). Culex quinquefasciatus holds historical significance; it was previously a subspecies of Culex pipiens, which is the first insect in which Wolbachia pipientis, an intracellular bacterial symbiont, was described (Hertig 1936, Ant et al. 2020).

Wolbachia is estimated to infect 40% to 60% of terrestrial arthropod species, making it one of Earth’s most prevalent symbiotic bacteria (Zug and Hammerstein 2012). This bacterium is maternally inherited and exerts diverse effects on its arthropod hosts. These effects include reproductive manipulations such as cytoplasmic incompatibility, parthenogenesis induction, and feminization of genetic males (Werren et al. 2008). Furthermore, Wolbachia can confer benefits to hosts, such as increased resistance to viral infections and a reduction in the capacity of mosquitoes to transmit pathogens like dengue and WNV (Caragata et al. 2021). A growing body of evidence suggests that Wolbachia density within host tissues is a key determinant of its functional impacts. Higher densities enhance the bacterium’s ability to mediate pathogen interference and reproductive manipulations, while lower densities reduce these effects (Ross et al. 2019).

The density and stability of Wolbachia in arthropod hosts is highly sensitive to environmental temperature. Temperatures exceeding 30 to 32 °C have been consistently shown to reduce Wolbachia density in mites, parasitoids, and mosquito species (Mouton et al. 2007, Wiwatanaratanabutr and Kittayapong 2009, Gharabigloozare and Bleidorn 2022). For instance, in the mosquito Aedes aegypti, elevated temperatures decrease Wolbachia levels, impairing its ability to block dengue virus replication (Ulrich et al. 2016, Ross et al. 2020). Studies on mosquito larvae and adults reveal a consistent decline in Wolbachia density under thermal stress (Farnesi et al. 2009, Ross et al. 2017, Tokash-Peters et al. 2022), yet their remains a critical gap, in that research on temperature effects on Wolbachia during the embryonic stage is limited, despite eggs being the obligate channel for vertical transmission of Wolbachia (Mouton et al. 2006). Evidence also suggests that cyclic or fluctuating regimes can produce more pronounced effects on Wolbachia than equivalent mean constant temperatures, because diurnal peaks intermittently push Wolbachia above its thermal tolerance and recovery between temperature peaks is incomplete (Ross et al. 2017, Mancini et al. 2021).

Studies have shown that mosquito embryos have specific temperature tolerances. Optimal development usually happens at mid-range temperatures, roughly 20 to 30 °C; however, temperatures that are too low or too high may slow down or stop embryogenesis, and can also reduce hatching success, and may lower survival rates (Farnesi et al. 2009, Moser et al. 2023). For Cx. quinquefasciatus, eggs could hatch at 15 °C and remain viable up to 40 °C, with optimal viability around 32 °C; nevertheless, development times decreased as temperatures increased, and mortality increased sharply above ∼34 °C (Moser et al. 2023). Because Wolbachia must persist in the embryo through this thermally sensitive developmental window for maternal transmission to succeed, embryos represent a potential bottleneck for symbiont transmission under heatwave conditions.

Additional studies indicate that high temperatures can reduce Wolbachia density in mosquitoes, but the specific thresholds and biological implications remain unclear (Wiwatanaratanabutr and Kittayapong 2009, Ulrich et al. 2016, Novakova et al. 2017, Ross et al. 2017, Ross et al. 2020, Mancini et al. 2021). This sensitivity not only can affect the stability of Wolbachia infections in mosquito populations but also can alter the bacterium’s ability to manipulate reproduction through mechanisms such as cytoplasmic incompatibility.

A decrease in Wolbachia density can have profound consequences for pathogen transmission dynamics. For example, low Wolbachia levels in Aedes aegypti have been linked to increased replication of dengue and chikungunya viruses (Ross et al. 2020). Similarly, in the Culex pipiens complex, reduced Wolbachia density in adult stages under thermal stress has been associated with higher WNV titers in infected individuals, enhancing their vector competence (Novakova et al. 2017). As global temperatures rise, the thermal sensitivity of Wolbachia may exacerbate disease transmission risks, particularly in regions experiencing extreme heat waves. A wider understanding of Wolbachia effects on Culex mosquitoes under temperature stress has been studied in the adult stages (Tokash-Peters et al. 2022); however, the early developmental stages in the mosquito life cycle, such as the embryonic stages, remain unclear.

Temperature can reduce Wolbachia density in reproductive tissues (Ross et al. 2017), impair maternal transmission, and even lead to complete loss of infection in embryos. Wolbachia infection in embryos has been assessed in other insect models, such as Drosophila melanogaster (Veneti et al. 2004), and Trichogramma dendrolimi and T. pretiosum (Zhou et al. 2023), yet less is known about its impacts in mosquito embryos. In Drosophila melanogaster, Wolbachia localization and density in the embryo govern subsequent germline colonization (Veneti et al. 2004, Serbus et al. 2011); in Trichogramma parasitoids, posterior concentration of Wolbachia during early embryogenesis determines adult tissue tropism (Zhou et al. 2023). Whether comparable embryonic dynamics operate in mosquitoes, and whether they are thermally robust, has not been directly tested. Quantifying Wolbachia density in mosquito embryos under realistic cyclic heat exposure is therefore a necessary first step to begin to consider how natural Wolbachia–mosquito associations will respond to climate change.

With global temperatures increasing due to climate change, understanding how thermal stress impacts Wolbachia density across mosquito developmental stages, including in embryos, is crucial.

Culex quinquefasciatus has a broad geographic distribution, and ambient air temperatures fluctuate daily up to 40 °C or higher during summer months throughout much of its range, including in the Central Valley of California (Hernandez et al. 2019). The objective of this study is to investigate whether high fluctuating environmental temperatures reduce Wolbachia density in the embryonic stage of Culex quinquefasciatus mosquitoes. Ultimately, this will contribute to our understanding of how increasing temperatures could disrupt Wolbachia densities in the embryo life stage. The outcome has implications for natural virus transmission cycles and for vector-mediated disease control strategies as well.

Methods

Mosquito Colonies

A Culex quinquefasciatus mosquito colony was established from parental mosquito adults from San Joaquin County Mosquito Abatement and Vector Control, Stockton, California. The colony originated from the region and has been maintained under laboratory conditions for approximately 100 generations. Wolbachia from Cx. quinquefasciatus in this region has been identified as wPip (Torres et al. 2020). Mosquitoes were maintained under laboratory conditions (25 ± 2 °C, 30% ± 5% RH, 16L:8D). Immature stages were raised with 250 to 300 larvae per cup, using dechlorinated water, and larvae were fed by adding 5 ml of 5% oat solution on days 0 and 9. Adults were fed with a 10% sugar solution ad libitum, and females were provided with chicken blood weekly for egg production to occur.

Wolbachia-Free Cx. quinquefasciatus Colony

A Wolbachia-free Cx. quinquefasciatus line was generated by tetracycline treatment (Dobson et al. 2004). Ten Cx. quinquefasciatus egg rafts were placed in individual 473-ml plastic cups containing 0.1 mg/ml tetracycline hydrochloride solution (Gibco Lot: M19l001, Life Technologies Corporation, Carlsbad, CA, USA), allowed to hatch, and reared under the conditions described above for the regular colony. Emerged adults were fed a 10% sugar solution containing tetracycline hydrochloride solution at a final concentration of 0.05 mg/ml ad libitum. Tetracycline can have transient effects on mosquito fitness (Ballard and Melvin 2007). For this reason, mosquitoes were maintained under antibiotic-free conditions for 2 further generations before use as controls, which both allows partial recovery of fitness and limits the probability of residual Wolbachia persistence. Complete Wolbachia clearance was verified by wsp qPCR and by FISH using the same protocol described below. Samples were retained as Wolbachia-free controls for the experiments and stored in 80% ethanol at 4 °C until use.

Embryo Developmental Stage Determination

To understand the developmental stages of Cx. quinquefasciatus embryos for later standardization of embryos in experiments, embryo development was examined under 2 different temperature regimes, a control of 25 °C and an elevated cyclical temperature regime of 25 to 40 °C. Egg rafts were exposed to a cyclical temperature regime that mimicked the naturally occurring daily summer temperature fluctuation in the Central Valley of California, where the Cx. quinquefasciatus colony originated. The experimental temperature regime began at 25 °C in the morning and increased to 40 °C in the afternoon, then decreased again to 25 °C in the early evening (Fig. 1). The control kept the eggs at 25 °C and this was used as a comparison to examine egg developmental stages (Fig. 1). The goal was to determine the number of hours for embryos to reach full development (but not hatching) at each temperature, so that embryos of a similar developmental stage could be used for comparison in experiments. A cyclical temperature regime with a high temperature of 40 °C was chosen because summer ambient air temperatures in the range of this species can increase up to approximately 40 °C during the day.

Fig. 1.

Experimental temperature conditions for the mosquito embryos of Culex quinquefasciatus used for FISH and qPCR experiments

Open in a new tab

Temperature stress profile for Culex quinquefasciatus embryos used in FISH and qPCR experiments at the control 25 °C, and the elevated cyclic temperature regime of 25 to 40 °C.

Newly oviposited egg rafts (0 to 1 h post-oviposition, HPO) were collected from the colony. Five egg rafts were placed at 25 °C and 5 additional egg rafts at the cyclical temperature treatment, 25 to 40 °C. Each egg raft has approximately 250 eggs (Kim et al. 2021). Every 2 h, an egg subsample was collected from each egg raft (5 eggs per time point, per temperature treatment) to check the egg maturation stage accordingly using a light microscope. This process continued until the eggs hatched. The maturation scale in hours for the mosquito embryonic development process, described by Juhn and James (2012), was used as a comparative measure. At each time point, the mean (± SE) percent egg development was determined. Once embryos reached a mean of 80% development, the hours since oviposition were noted and compared between the temperature treatments. The 80% developmental threshold is considered fully developed but not hatched (Juhn and James 2012). A Mann–Whitney U test was used to compare the time until full embryonic development between the control 25 °C and the cyclic temperature regime, 25 to 40 °C.

Wolbachia Density in Cx. quinquefasciatus Embryos Exposed to Cyclical High Temperature

Embryo temperature exposure

Egg rafts (each ∼200 to 250 eggs) were collected from the mosquito colony 24 h post-oviposition (HPO), and 50 rafts were exposed to each temperature regime (50 rafts at 25 °C, 50 rafts at 25 to 40 °C). This enabled standardization during the developmental stage of the egg rafts (embryos) for later comparison. Using incubator chambers (IVYX Scientific 25L incubator, Carolina Biological Supply, Burlington, NC, USA), 10 replicates of 5 egg rafts (50 rafts per temperature treatment) were put in a 150-ml plastic cup with water and exposed to the fluctuating temperature regime from 25 °C up to 40 °C for 15 continuous hours. These egg rafts were later used for the FISH and qPCR experiments, described below.

The 50 replicate egg rafts from both the control (25 °C) and the cyclic temperature regime (25 to 40 °C) (Fig. 1) were divided in half; the first half was used for Wolbachia detection through the FISH method, and the second half was used for Wolbachia molecular detection using the qPCR technique. Egg rafts were collected in 80% ethanol and stored at 4 °C until they were processed.

Preparing embryos for FISH

As described above, 50 egg rafts were subjected to each temperature treatment (25 °C, 25 to 40 °C cyclic). An embryo was removed from each egg raft and prepared for FISH (50 embryos/1 per raft/per temperature = 50 embryos per temperature treatment). Embryos were prepared for FISH following the adapted protocol described by Juhn and James (2012). Briefly, approximately 50 eggs were dechorionated by exposing them to 5 ml hypochlorite solution (1:3, 5.25% sodium hypochlorite: distilled water) for 72 s and then washed by immersion in pure water. Eggs were held 72 s in hypochlorite since it provided complete chorion removal without visible damage to the vitelline membrane. Using 5 ml of 99% heptane, water was removed, and the whole eggs were put in 5 ml fresh fixation solution (0.4 ml 37% formaldehyde) in 4.6 ml PBT (phosphate-buffered saline [PBS], pH 7.4, 0.1% Tween 20) for 30 min. After that, the fixation solution was removed, and the eggs were washed with pure water 3 times. Subsequently, 5 ml heptane and 5 ml 99.8% methanol were added, swirled twice, and discarded. Finally, 5 ml of methanol was added.

After fixation, the chorion was peeled from the egg to leave just the embryo. For peeling, eggs were transferred to a slide with double-sided tape and allowed to adhere to the tape surface, and several drops of ethanol and pure water were added. Using a minuten pin, the cracked endochorion was peeled. After removing the chorion, the embryos were transferred to 99.5% ethanol for storage until the yolk removal step. Clarification of embryo yolk was done using 99% P-xylene/99.5% ethanol (9:1) at 22 to 24 °C for 75 min, then embryos were washed using 99% methanol. Finally, samples were equilibrated by washing with 5 ml 99% methanol/formaldehyde fixation solution (1:1). The additional fixation stabilizes embryonic cytoarchitecture and improves probe retention (Juhn and James 2012).

Embryos were fixed in a freshly prepared fixation solution (described above) at room temperature for 30 min. The fixation solution was decanted and then washed using PBT. Then, protein digestion was followed with 0.01 mg/ml Proteinase K/PBT for 5 min at room temperature. Afterwards, the digestion solution was removed, and 3 washes were performed with ice-cold PBT. A soak in fixation solution was allowed for 30 min, and then 2 final PBT washes were made.

FISH and microscopy

A DNA probe targeting the wsp gene and labeled at the 3′ end with rhodamine (W3 5′-AACCGACCCTATCCCTTCGAATA-3′) (ABNOVA, Taipai, Taiwan) was used to localize Wolbachia in mosquito embryo tissues following the workflow of Juhn and James (2012). The probe was diluted to 1× with FISH hybridization buffer. Embryos were transferred to a microfuge tube, and 10 μl probe solution was added. Hybridization was carried out for 8 h at 37 °C inside a chamber described above. After incubation, the probe solution was removed, and samples were washed with 2× SSB buffer for 5 min at room temperature. The 2× SSB buffer was removed, and 0.4× SSB buffer/0.3% NP-40 was added at 73 to 75 °C for 1–2 min. The 0.4× SSB buffer/0.3% NP-40 was removed, and the 2× SSB buffer was added again at room temperature for 1 min. Subsequently, embryos were transferred to microfuge tubes, and 70% glycerol/H2O was pipetted on top of the samples. Glycerol was allowed to permeate the sample tissue. Microscope slides were prepared by placing 2 pieces of clear tape parallel to each other to serve as a narrow channel for embryos. Embryos were picked up one by one and placed along the length of the channel, positioning each sample for anterior/posterior and dorsal/ventral alignments. After positioning, the excess glycerol solution was removed, and 10 μl UltraCruz mounting medium solution containing DAPI (sc-24941, Santa Cruz Biotechnology, 1.5 μg/ml) was added to the channel tape containing the aligned embryos, and a cover slip was placed over the channel. DAPI stains the cell nuclei blue to provide contrast with the DNA probe. The whole mount was affixed with transparent nail polish.

A confocal microscope (Zeiss, LSM 880) at the Image and Microscopy Facilities (IMF) at the University of California, Merced, was used for imaging embryos. Slides selected and used to process images followed pre-defined criteria: each embryo retained for quantification had intact morphology after the FISH protocol, detectable DAPI nuclear staining throughout the embryo, no obvious tissue damage from chorion removal, and a confirmed anterior/posterior–dorsal/ventral orientation. Acquisition settings (laser power, gain, pinhole, pixel dwell time, z-step) were held constant across all samples. Eight samples from each temperature regime (25 °C, 25 to 40 °C) met the criteria.

Fluorescence intensity was quantified in FIJI/ImageJ 1.54f (Schindelin et al. 2012, Schneider et al. 2012) using a standardized integrated-density workflow. Each multi-channel confocal stack was projected (maximum intensity) and split into individual channels; the DAPI channel was used to define the embryo region of interest (ROI) through Otsu thresholding, binary mask generation, and morphological clean-up. The resulting ROI was saved and applied to the Wolbachia (rhodamine) channel to measure signal within the same embryonic region. Area, mean gray value, and integrated fluorescence density (the product of mean fluorescence intensity and ROI area) were recorded. Integrated fluorescence density was compared between treatments (n = 8 per treatment) using a 2-tailed Welch’s t-test in R 4.3.2 (R Core Team 2024) via RStudio (Posit Team 2025), with α  =  0.05. Normality (Shapiro–Wilk) and homogeneity of variance (Levene’s test) were checked prior to the t-test.

Wolbachia Relative Quantification

Egg rafts subjected to the temperature regime described above were used for relative quantification of Wolbachia density in Cx. quinquefasciatus embryos. For each temperature treatment, 50 egg rafts were used; for each egg raft, half the egg raft was used as the sample Genomic DNA was extracted from egg rafts using the Qiagen DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA, USA), following the manufacturer’s protocol for tissue extraction with an overnight incubation at 65 °C (Qiagen 2023); the elevated incubation temperature (65 °C) improved digestion of the chitinous chorion and yolk-rich embryonic tissue and is supported by manufacturer-permitted protocol modifications for difficult-to-lyse samples (Qiagen 2023). Extracted DNA was stored at −20 °C. The DNA quantity was measured using the Qubit dsDNA HS Assay kit (Life Technologies, Carlsbad, CA, USA). The quantity of DNA in the samples was concentrated using the Eppendorf Vacufuge and standardized to 15 ± 3 ng/µl. The narrow concentration range was selected because preliminary qPCR runs showed Ct stability within this window.

Wolbachia presence in egg rafts was determined by amplicon detection of the general Wolbachia surface protein (wsp) using the primers (F 5′-GCATTTGGTTAYAAAATGGACGA-3′, R 5′-GGAGTGATAGGCATATCTTCAAT-3′) via qPCR. PowerUP SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) was used following the recommended manufacturer’s conditions. The qPCR cycling conditions were as follows: 95 °C for 10 min and 40 cycles of 15 s at 95 °C and 1 min at 60 °C followed by a melt-curve stage (60 to 95 °C, 0.3 °C increments). Each sample was analyzed in triplicate, and a non-template control was included. The qPCR assays were run on Applied Biosystems 5700 Fast Real-time PCR (Applied Biosystems, Foster City, CA, USA). Relative density was determined by measuring the signal amplifications of the wsp gene and Ribosomal protein S3 gene (RpS3) (RpS3: 70F 5′-AGCGTGCCAAGTCGATGAG-3′, R 5′-ACGTACTCGTTGCACGGATCTC-3′), which was used as a reference (housekeeping) gene and has been found to be stable and appropriate to use in insects under thermal stress (Livak and Schmittgen 2001, Torres et al. 2020, Sellamuthu et al. 2021). Samples were compared, and the data were analyzed post-run using the ΔΔCt method (Livak and Schmittgen 2001). Statistical comparison of ΔΔCt values between treatments was performed in R 4.3.2 (R Core Team 2024) using a Mann–Whitney U test. Negative controls included the Wolbachia-free Cx. quinquefasciatus colony, which was treated with the antibiotic tetracycline, and Aedes aegypti.

Results

Culex quinquefasciatus Embryonic Developmental Stage Determination

The elevated cyclical temperature treatment (25 to 40 °C) affected the embryonic development rate in the Cx. quinquefasciatus eggs, accelerating larval development by 8 h compared with the 25 °C temperature treatment (Mann–Whitney U 2-tailed test, U = 0, P = 0.009) (Supplementary Table S1, Fig. 2A to E). While embryos exposed to the fluctuating temperature regime up to 40 °C required 26 h to mature completely (100%), those at 25 °C took approximately 34 h (Supplementary Table S1, Fig. 2E).

Fig. 2.

Embryonic development stages for Culex quinquefasciatus exposed to 25 °C and 25-40°C at 14- and 26-h post-oviposition (HPO)

Open in a new tab

Embryonic development stages for Culex quinquefasciatus (A, C) exposed to 25 °C at 14- and 26-h post-oviposition (HPO) and (B, D) exposed to cyclic 25 to 40 °C at 14 and 26 HPO. Embryogenesis is assumed to be complete by 26 h, evidenced by the formation of the hatching spine, the full differentiation of head, thorax, and abdominal segments, and the presence of eye spots. Score was assigned according the embryonic-maturation scale of Juhn and James (2012). E). The mean (± SE) percent egg development at 25 °C and the cyclical elevated 25 to 40 °C temperature treatments (see Supplementary Table S1).

FISH Detection of Wolbachia in the Cx. quinquefasciatus Embryos

Eggs used for experiments were standardized at a developmental stage of approximately 70% maturation; they were 26 h post-oviposition (HPO) when exposed to 25 °C and the cycling temperatures 25 to 40 °C (Fig. 2E). FISH labelling with the wsp-targeted W3 probe produced a clear Wolbachia signal in Cx. quinquefasciatus embryos at both thermal treatments and no detectable signal in the Wolbachia-free, tetracycline-treated control embryos (Fig. 2A), confirming probe specificity. DAPI counter-staining produced the expected blue nuclear signal in all embryos (Fig. 2A).

No bacterial signal was detected for the Wolbachia-free Cx. quinquefasciatus embryos treated with tetracycline (negative controls) (Fig. 3A). The embryo cell nuclei are shown to be stained blue with DAPI (Fig. 3A). In general, Wolbachia bacteria were localized along the central part of the Cx. quinquefasciatus embryos, shown in red, and consistently concentrated in the germ band cells at both temperature treatments (Fig. 3B).

Fig. 3.

Wolbachia detection in Culex quinquefasciatus embryos. The left shows a control which is Wolbachia free, the right image shows red fluorescence where Wolbachia is present.

Open in a new tab

Wolbachia detection in Culex quinquefasciatus embryos. A) Embryo treated with tetracycline and Wolbachia-free, B) Embryos infected with Wolbachia. Red fluorescent emission for Wolbachia, blue fluorescence emission from DAPI indicating the embryo cells’ nuclei.

High Temperature and Wolbachia Density in Cx. quinquefasciatus Embryos

Confocal images of FISH-labeled embryos showed reduced Wolbachia fluorescence in embryos exposed to the cyclic 25 to 40 °C regime compared with embryos held at constant 25 °C (Fig. 4A and B). The qualitative pattern of lower signal under the cyclic regime was consistent across the 8 embryos imaged per treatment. Quantitative comparison of integrated fluorescence density confirmed the visual pattern. The comparison of the integrated fluorescence density measured in Cx. quinquefasciatus embryos at both temperatures showed a significant difference in Wolbachia density (t = 2.21, df = 14, P = 0.04), with lower bacteria density when temperature fluctuated up to 40 °C. Both data sets followed a normal distribution (Shapiro-Wilk test, P = 0.901 for 25 °C, P = 0.783 for 40 °C) and exhibited homogeneity of variance (Levene’s test, P = 0.41) (Supplementary Table S2).

Fig. 4.

Integrated fluorescence density for Wolbachia detection in Culex quinquefasciatus embryos. Image on left at 25 °C, image on right at cyclic temperature of 25 °C-40°C

Open in a new tab

Integrated fluorescence density for Wolbachia detection in Culex quinquefasciatus embryos (A) exposed to 25 °C and (B) exposed to cyclic temperatures 25 to 40 °C.

The qPCR-based estimate of relative Wolbachia density found a reduction in density at the elevated cyclic temperature. Relative density was calculated using ΔΔCt with RpS3 as the reference gene and the constant 25 °C treatment as the calibrator (Livak and Schmittgen 2001). Data were not normally distributed, so a 2-tailed Mann–Whitney U test was performed. Relative Wolbachia density was significantly lower in embryos exposed to the cyclic 25 to 40 °C regime than in embryos held at constant 25 °C (Mann–Whitney U 2-tailed test, U = 496, P = 0.021, n = 41 egg rafts at constant 25 °C, n = 35 egg rafts at cyclic 25 to 40 °C) (Fig. 5, Supplementary Table S3). The mean fold reduction (mean 25 °C ÷ mean 40 °C) was 3.4. Both negative controls, tetracycline-treated Wolbachia-free Cx. quinquefasciatus and Aedes aegypti, returned no detectable wsp amplification, confirming that the qPCR signal in the experimental samples reflects wsp from Cx. quinquefasciatus-associated Wolbachia.

Fig. 5.

Relative quantification of Wolbachia in Culex quinquefasciatus embryos using wsp gene and RpS3 housekeeping gene

Open in a new tab

Relative quantification of Wolbachia in Culex quinquefasciatus embryos using wsp gene and RpS3 housekeeping gene, with embryos exposed at 25 °C and cyclic 25 to 40 °C. Mann–Whitney U 2-tailed test, U = 496, P = 0.021.

Discussion

This study shows that elevated temperatures, with daily thermal exposure ranging from 25 to 40 °C, significantly decrease Wolbachia density in Cx. quinquefasciatus embryos. Using both FISH for visual detection of Wolbachia density, and qPCR for relative quantification, a consistent decrease in Wolbachia load in the embryonic stage was observed under cyclical heat-stress conditions. These findings are especially relevant given the potential role of this bacterium in modulating vector competence for West Nile virus (WNV), suggesting that thermal stress during embryonic development could impair Wolbachia’s ability to influence viral transmission in natural mosquito populations.

The observed acceleration of embryonic development at cyclic temperatures up to 40 °C compared to 25 °C highlights the sensitivity of Cx. quinquefasciatus to temperature variations. The acceleration of embryonic development under the cyclic 25 to 40 °C regime, approximately 8 h earlier, is consistent with the established thermal biology of mosquito embryos, in which higher temperatures shorten embryogenesis up to a species-specific upper limit (Farnesi et al. 2009, Reinhold et al. 2018, Moser et al. 2023). Such accelerated development may have ecological consequences, potentially altering population dynamics and vector capacity in warmer climates or during heat waves. Such rapid growth may affect the quality and viability of larvae, as has been suggested in other mosquito species (Reinhold et al. 2018); however, larval viability and other fitness trade-offs were not assessed in the current study and warrant further study.

The Wolbachia signal in Cx. quinquefasciatus embryos was concentrated in the central embryonic region corresponding to the germ band at both temperatures, a localization pattern consistent with previous descriptions in Drosophila (Serbus et al. 2011) and Trichogramma embryos (Zhou et al. 2023). The lower fluorescence intensity at the cyclic 25 to 40 °C regime is consistent with a reduction in bacterial density; fluorescence intensity is also influenced by probe penetration, staining efficiency, and microscope acquisition settings. The convergent reduction in Wolbachia density at the cyclic 25 to 40 °C regime, detected by both FISH-based integrated fluorescence density and qPCR-based ΔΔCt, is consistent with the broader literature on thermal sensitivity of Wolbachia in arthropod hosts (Mouton et al. 2007, Wiwatanaratanabutr and Kittayapong 2009, Ulrich et al. 2016, Ross et al. 2017).

The magnitude of temperature increase could impact the reduction in Wolbachia density. For example, Tokash-Peters et al. (2022) found that elevated temperatures (28 to 30 °C compared to 23 °C) did not significantly influence Wolbachia abundance within the same generation of adult Cx. quinquefasciatus; however, across successive generations, there was a notable decrease in Wolbachia load when comparing identical temperature conditions. In addition, their findings were for adult mosquitoes, while embryos may have different temperature sensitivities. It is possible that the temperature parameters examined by these authors were insufficient to induce a reduction in bacterial load. However, increasing the temperature fluctuation up to 40 °C has been shown to produce significant effects, as evidenced in the current study, particularly when considering the sensitive mosquito developmental stage (embryos) that was evaluated here. Novakova et al. (2017) found that there was a correlated decrease in the relative abundance of Wolbachia in field-collected adult Cx. pipiens during periods of elevated seasonal temperatures, highlighting high temperatures as one of the important abiotic factors that diminishes Wolbachia levels and potentially heightens WNV susceptibility in Culex mosquitoes. Others found that Wolbachia abundance in field-collected adult Cx. pipiens is inversely related to environmental temperature, and higher temperature correlates with lower Wolbachia levels and higher WNV prevalence (Garrigós et al. 2023). The current study adds to knowledge of temperature impacts on Wolbachia density in an additional mosquito life stage (embryos), which has not been previously investigated.

The potential thermal modulation of Wolbachia density could have significant consequences for symbiotic efficacy and mosquito vector competence. Wolbachia is known to influence mosquito biology through cytoplasmic incompatibility, pathogen blocking, and fitness effects (Walker et al. 2011, Zug and Hammerstein 2012). A reduction in bacterial density at high temperatures could compromise these symbiotic benefits, thereby altering disease transmission dynamics. For example, the thermal sensitivity of Wolbachia in Aedes aegypti has been implicated in the diminished efficacy of pathogen-blocking traits under field conditions (Ross et al. 2017). These findings suggest that climate warming may compromise natural Wolbachia-mediated pathogen suppression, which may occur in other mosquito species as well, and could be considered with regard to timing and strain selection for Wolbachia-based vector control programs. However, previous studies focus on Ae. aegypti, and further study of the thermal impacts of Wolbachia in the Cx. pipiens complex under field conditions will be required. Thermal sensitivities established in Aedes infected with wMel may not transfer directly to Culex spp. with a different Wolbachia strain (wPip), given known differences in and thermal tolerances (Mancini et al. 2021).

The interplay between temperature, host development, and Wolbachia density is likely to have broader ecological and evolutionary implications. Climate change, characterized by rising temperatures and an increased frequency of extreme heat events, may disrupt host-symbiont interactions in mosquito populations, with cascading effects on vector ecology and epidemiology (Mordecai et al. 2019). While our findings are derived from controlled laboratory conditions and restricted to the embryonic stage, they highlight the sensitivity of vertically transmitted symbionts to early-life thermal extremes.

This study provides valuable insights into temperature impacts on Wolbachia density in embryos by examining fluctuating temperatures up to 40 °C. There are several limitations which warrant consideration. First, the experimental conditions, even with cycling temperatures, may not fully replicate natural thermal fluctuations experienced by mosquitoes in the field. For example, field conditions are more complex, with factors such as vegetation to moderate temperature peaks. A study examining field-collected Cx. quinquefasciatus life stages through seasonal temperature peaks would provide insight into how fluctuating temperatures impact the level of Wolbachia, especially through several generations in the field and during the peak vector transmission season. Second, the focus on embryonic stages can be expanded to other life stages to examine how temperature stress and Wolbachia dynamics continue to manifest during later life stages and in Culex spp. of mosquitoes. Also of interest is the question of whether Wolbachia density would increase once temperatures cool; this would contribute to a better understanding of ecological dynamics of the system. Future studies should consider longitudinal tracking of Wolbachia density and host fitness across the mosquito life cycle. Additionally, absolute quantification of Wolbachia copy number would further clarify the magnitude of thermal effects and strengthen the interpretation of density-dependent responses.

This study underscores the nuanced effects of cyclical high temperatures up to 40 °C on Cx. quinquefasciatus embryonic development and Wolbachia density. At elevated cyclic temperatures, Cx. quinquefasciatus embryos demonstrated quicker developmental time, and both FISH and qPCR found a reduction in Wolbachia density. This study highlights the vulnerability of the Cx. quinquefasciatus and Wolbachia host-symbiont system to thermal stress. These findings could have implications for transmission of vector-borne disease and potentially vector-mediated disease control strategies under changing climatic conditions, and should be further investigated.

Supplementary Material

ieag079_Supplementary_Data
ieag079_supplementary_data.zip (28.5KB, zip)

Acknowledgements

The authors thank Abril Landeros of University of Guadalajara for the support in the lab procedures, and the Image and Microscopy Facility (IMF) at the University of California for the accessories for the image analysis. Special thanks to the University of California Merced for their financial support of this research project. We thank Jose Luis Ramirez of USDA ARS Crop Protection Research in Peoria Illinois for helpful discussions.

Contributor Information

Jovany Barajas, Environmental Systems, University of California Merced, Merced, CA, 95343, USA.

Shaoming Huang, San Joaquin County Mosquito and Vector Control District, Stockton, CA, 95206, USA.

Fabian Gonzalez, Public Health, University of California Merced, Merced, CA, 95343, USA.

Andrea Joyce, Public Health, University of California Merced, Merced, CA, 95343, USA.

Author Contributions

Jovany Barajas (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Shaoming Huang (Project administration [equal], Resources [equal], Writing—original draft [equal], Writing—review & editing [equal]), Fabian Gonzalez (Data curation [equal], Formal analysis [equal], Investigation [equal]), and Andrea Joyce (Conceptualization [equal], Formal analysis [equal], Funding acquisition [equal], Methodology [equal], Project administration [equal], Resources [equal], Supervision [equal], Validation [equal], Writing—original draft [equal], Writing—review & editing [equal])

Supplementary Material

Supplementary material is available at Journal of Insect Science online.

Funding

None declared.

Conflicts of Interest

None declared.

References

  1. Ant TH, Herd C, Louis F, et al.  2020. Wolbachia transinfections in Culex quinquefasciatus generate cytoplasmic incompatibility. Insect Mol. Biol. 29:1–8. 10.1111/imb.12604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballard JWO, Melvin RG.  2007. Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila. Insect Mol. Biol. 16:799–802. 10.1111/j.1365-2583.2007.00760.x [DOI] [PubMed] [Google Scholar]
  3. Bartholomay LC, Waterhouse RM, Mayhew GF, et al.  2010. Pathogenomics of Culex quinquefasciatus and meta-analysis of infection responses to diverse pathogens. Science. 330:88–90. 10.1126/science.1193162 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caragata EP, Dutra HLC, Sucupira PHF, et al.  2021. Wolbachia as translational science: controlling mosquito-borne pathogens. Trends Parasitol. 37:1050–1067. 10.1016/j.pt.2021.06.007 [DOI] [PubMed] [Google Scholar]
  5. Dobson SL, Rattanadechakul W, Marsland EJ.  2004. Fitness advantage and cytoplasmic incompatibility in Wolbachia single- and superinfected Aedes albopictus. Heredity. (Edinb)  93:135–142. 10.1038/sj.hdy.6800458 [DOI] [PubMed] [Google Scholar]
  6. Farnesi LC, Martins AJ, Valle D, et al.  2009. Embryonic development of Aedes aegypti (Diptera: Culicidae): influence of different constant temperatures. Mem. Inst. Oswaldo Cruz. 104:124–126. 10.1590/s0074-02762009000100020 [DOI] [PubMed] [Google Scholar]
  7. Garrigós M, Garrido M, Panisse G, et al.  2023. Interactions between West Nile virus and the microbiota of Culex pipiens vectors: a literature review. Pathogens.  12:1287. 10.3390/pathogens12111287 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gharabigloozare Y, Bleidorn C.  2022. Effect of high temperature on Wolbachia density and impact on cytoplasmic incompatibility in confused flour beetle, Tribolium confusum (Coleoptera: Tenebrionidae). BMC Res. Notes.  15:240. 10.1186/s13104-022-06123-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hernandez E, Torres R, Joyce AL.  2019. Environmental and sociological factors associated with the incidence of West Nile virus cases in the Northern San Joaquin Valley of California, 2011–2015. Vector Borne Zoonotic Dis. 19:851–858. 10.1089/vbz.2019.2437 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hertig M.  1936. The Rickettsia, Wolbachia pipientis (gen. et sp. n.). Parasitology.  28:453–486. [Google Scholar]
  11. Huang S, Smith DJ, Molaei G, et al.  2013. Prevalence of Dirofilaria immitis (Spirurida: Onchocercidae) infection in Aedes, Culex, and Culiseta mosquitoes from North San Joaquin Valley, CA. J. Med. Entomol. 50:1315–1323. 10.1603/me13111 [DOI] [PubMed] [Google Scholar]
  12. Iwamura T, Guzman-Holst A, Murray KA.  2020. Accelerating invasion potential of disease vector Aedes aegypti under climate change. Nat. Commun. 11:2130. 10.1038/s41467-020-16010-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Juhn J, James AA.  2012. Hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes. J. Vis. Exp. 64:3709. 10.3791/3709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim DY, Leepasert T, Bangs MJ, et al.  2021. Evaluation of mosquito attractant candidates using a high-throughput screening system for Aedes aegypti (L.), Culex quinquefasciatus Say. and Anopheles minimus Theobald (Diptera: Culicidae). Insects. 12:528. 10.3390/insects12060528 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kramer LD, Styer LM, Ebel GD.  2008. A global perspective on the epidemiology of West Nile virus. Annu. Rev. Entomol. 53:61–81. 10.1146/annurev.ento.53.103106.093258 [DOI] [PubMed] [Google Scholar]
  16. Ledesma N, Harrington L.  2011. Mosquito vectors of dog heartworm in the United States: vector status and factors influencing transmission efficiency. Top. Companion Anim. Med. 26:178–185. 10.1053/j.tcam.2011.09.005 [DOI] [PubMed] [Google Scholar]
  17. Livak KJ, Schmittgen TD.  2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 25:402–408. 10.1006/meth.2001.1262 [DOI] [PubMed] [Google Scholar]
  18. Mancini MV, Ant TH, Herd CS, et al.  2021. High temperature cycles result in maternal transmission and dengue infection differences between Wolbachia strains in Aedes aegypti. mBio. 12:e00250-21. 10.1128/mBio.00250-21 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mordecai EA, Caldwell JM, Grossman MK, et al.  2019. Thermal biology of mosquito-borne disease. Ecol. Lett. 22:1690–1708. 10.1111/ele.13335 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moser SK, Barnard M, Frantz RM, et al.  2023. Scoping review of Culex mosquito life history trait heterogeneity in response to temperature. Parasit. Vectors. 16:200. 10.1186/s13071-023-05792-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mouton L, Henri H, Bouletreau M, et al.  2006. Effect of temperature on Wolbachia density and impact on cytoplasmic incompatibility. Parasitology. 132:49–56. 10.1017/S0031182005008723 [DOI] [PubMed] [Google Scholar]
  22. Mouton L, Henri H, Charif D, et al.  2007. Interaction between host genotype and environmental conditions affects bacterial density in Wolbachia symbiosis. Biol. Lett. 3:210–213. 10.1098/rsbl.2006.0590 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Novakova E, Woodhams DC, Rodríguez-Ruano SM, et al.  2017. Mosquito microbiome dynamics, a background for prevalence and seasonality of West Nile virus. Fron.t Microbiol.  8:526. 10.3389/fmicb.2017.00526 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Posit Team. 2025. RStudio: integrated development environment for R. Posit software, PBC, Boston, MA.
  25. Qiagen. 2023. DNeasy blood & tissue handbook. Qiagen, Venlo, Netherlands. [Google Scholar]
  26. R Core Team. 2024. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/
  27. Rasgon JL, Scott TW.  2003. Wolbachia and cytoplasmic incompatibility in the California Culex pipiens mosquito species complex: parameter estimates and infection dynamics in natural. Genetics  165:2029–2038. 10.1093/genetics/165.4.2029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reinhold JM, Lazzari CR, Lahondère C.  2018. Effects of the environmental temperature on Aedes aegypti and Aedes albopictus mosquitoes: a review. Insects. 9:158. 10.3390/insects9040158 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ross PA, Axford JK, Yang Q, et al.  2020. Heatwaves cause fluctuations in wMel Wolbachia densities and frequencies in Aedes aegypti. PLoS Negl. Trop. Dis. 14:e0007958. 10.1371/journal.pntd.0007958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ross PA, Turelli M, Hoffmann AA.  2019. Evolutionary ecology of Wolbachia releases for disease control. Annu. Rev. Genet. 53:93–116. 10.1146/annurev-genet-112618-043609 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ross PA, Wiwatanaratanabutr I, Axford JK, et al.  2017. Wolbachia infections in Aedes aegypti differ markedly in their response to cyclical heat stress. PLoS Pathog. 13:e1006006. 10.1371/journal.ppat.1006006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Saarman N, Graybeal K, Seeley T, et al.  2025. Range expansion of Culex quinquefasciatus and Culex pipiens hybrids across mid-latitudes of North America. One Health. 21:101205. 10.1016/j.onehlt.2025.101205 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Salim KU, Álvarez FS, Chan-Golston AM, et al.  2024. Socioeconomic and environmental factors associated with dengue fever incidence in Guatemala: rising temperatures increase dengue risk. PLoS One. 19:e0308271. 10.1371/journal.pone.0308271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schindelin J, Arganda-Carreras I, Frise E, et al.  2012. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9:676–682. 10.1038/nmeth.2019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schneider CA, Rasband WS, Eliceiri KW.  2012. NIH Image to Image J: 25 years of image analysis. Nat. Methods.  9:671–675. 10.1038/nmeth.2089 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sellamuthu G, Amin S, Bílý J, et al.  2021. Reference gene selection for normalizing gene expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) under different experimental conditions. Front. Physiol. 12:752768. 10.3389/fphys.2021.752768 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Serbus LR, Ferreccio A, Zhukova M, et al.  2011. A feedback loop between Wolbachia and the Drosophila gurken mRNP complex influences Wolbachia titer. J. Cell Sci. 124:4299–4308. 10.1242/jcs.092510 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tokash-Peters AG, Jabon JD, Fung ME, et al.  2022. Trans-generational symbiont transmission reduced at high temperatures in a West Nile virus vector mosquito Culex quinquefasciatus. Front. Trop. Dis. 3:762132. 10.3389/fitd.2022.762132 [DOI] [Google Scholar]
  39. Torres R, Hernandez E, Flores V, et al.  2020. Wolbachia in mosquitoes from the Central Valley of California, USA. Parasit. Vectors. 13:558. 10.1186/s13071-020-04429-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ulrich JN, Beier JC, Devine GJ, et al.  2016. Heat sensitivity of wMel Wolbachia during Aedes aegypti development. PLoS Negl. Trop. Dis. 10:e0004873. 10.1371/journal.pntd.0004873 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Veneti Z, Clark ME, Karr TL, et al.  2004. Heads or tails: host-parasite interactions in the Drosophila-Wolbachia system. Appl. Environ. Microbiol. 70:5366–5372. 10.1128/AEM.70.9.5366-5372.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walker T, Johnson PH, Moreira LA, et al.  2011. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Nature. 476:450–453. 10.1038/nature10355 [DOI] [PubMed] [Google Scholar]
  43. Werren JH, Baldo L, Clark ME.  2008. Wolbachia: master manipulators of invertebrate biology. Nat. Rev. Microbiol. 6:741–751. 10.1038/nrmicro1969 [DOI] [PubMed] [Google Scholar]
  44. Wiwatanaratanabutr I, Kittayapong P.  2009. Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus. J. Invertebr. Pathol. 102:220–224. 10.1016/j.jip.2009.08.009 [DOI] [PubMed] [Google Scholar]
  45. Zhou JC, Dong QJ, Shang D, et al.  2023. Posterior concentration of Wolbachia during the early embryogenesis of the host dynamically shapes the tissue tropism of Wolbachia in host Trichogramma wasps. Front. Cell. Infect. Microbiol.  13:1198428. 10.3389/fcimb.2023.1198428 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zug R, Hammerstein P.  2012. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One. 7:e38544. 10.1371/journal.pone.0038544 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ieag079_Supplementary_Data
ieag079_supplementary_data.zip (28.5KB, zip)

Articles from Journal of Insect Science are provided here courtesy of University of Wisconsin Libraries

RESOURCES