Ae. albopictus and Ae. aegypti strains and rearing
Two Ae. albopictus strains and one Ae. aegypti strain were used in the experiments. SANG Ae. albopictus originated from wild-type individuals collected from Anguillara Sabazia (Rome) in 2006 and harbors wAlbA and wAlbB Wolbachia. ARwP Ae. albopictus was established in 2008 through the transinfection of Wolbachia-cured SANG individuals with wPip Wolbachia from Culex pipiens molestus [37] and is characterized by a bidirectional incompatibility pattern with wild-type Ae. albopictus [38]. Both lines were reared under laboratory conditions at the National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA Casaccia Research Center (Rome) and were periodically outcrossed with wild-type individuals from the same area to preserve genetic variability [39]. The Ae. aegypti line (New Orleans, LA 2011) was provided by the University of Camerino (Camerino, MC, Italy), where it had been laboratory-reared since 2014 and was not infected with Wolbachia.
Colonies were maintained by raising larvae to adulthood inside 1-l larval trays at a density of 1 larva/ml, provided with liquid food according to the methods described in a previous study [39]. Adult mosquitoes were maintained inside 40 × 40 × 40 cm cages at temperature of 28 ± 1 °C, relative humidity (RH) of 70% ± 10%, and 14:10-h light/dark cycle, and were supplied with water and 10% sucrose.
Blood meals were provided via anesthetized mice in agreement with the Bioethics Committee for Animal Experimentation in Biomedical Research and in accordance with procedures approved by the ENEA Bioethical Committee according to the EU directive 2010/63/EU. The mice belonged to a colony housed at CR ENEA Casaccia and maintained for experimentation based on the authorization no. 80/2017-PR released (on February 2, 2017) by the Italian Ministry of Health. Feeding of female mosquitoes on the blood of human hosts (i.e., the authors RM, EL, GL, and MC) during the experiments was also approved by the ENEA Bioethical Committee.
Radiation methods
Cohorts of Ae. albopictus and Ae. aegypti females belonging to the strains described above were irradiated with X-rays to enable comparison with sham-exposed individuals (pupae treated in a manner similar to the exposed pupae except for the X-ray exposure).
Aedes albopictus pupae were sexed mechanically using a specific sieving procedure described previously [16].
X-ray irradiation was performed using the Gilardoni CHF 320G X-ray generator (Gilardoni S.p.A.; Mandello del Lario, Lecco, Italy) operated at 250 kVp, and 15 mA, with filters of 2.0 mm of Al and 0.5 mm of Cu, furnished by the Physical Technologies for Security and Health Division of ENEA. Depending on the experiment and according to doses already tested for the radiation-based sterilization of the two species [9, 19, 21], Ae. albopictus pupae were subjected to 28, 35, and 45 Gy (dose rate: 0.868 ± 0.004 Gy/min, mean ± SD), while a single dose of 50 Gy was used to treat Ae. aegypti, as this dosage is known to fully inhibit egg production in the species [9]. Time of sample exposure was determined according to dose rate in order to obtain the pre-established doses. Doses were confirmed by monitoring the exposure with a PTW 7862 large-size plane-parallel transmission chamber connected to a PTW IQ4 electrometer. Groups of 100 female pupae were transferred to a Petri dish (d = 4 cm) at 36 ± 4 h of age (unless specified differently) and then transported to the irradiation facility. Immediately before commencement of irradiation, most of the residual water was removed using a glass pipette; irradiated pupae were then transferred to a larger water container to facilitate complete development and allow for adult emergence inside the experimental cages.
Survival, fecundity, and fertility in irradiated Ae. albopictus females
SANG or ARwP Ae. albopictus females irradiated at 28, 35, and 45 Gy, and untreated counterparts were allowed to emerge inside 30 × 30 × 30 cm plastic cages, which were checked for the presence of males that escaped the sexing procedure. Thirty virgin females and thirty untreated males—characterized by the same Wolbachia infection type—were placed in each cage.
These cages were used to monitor survival, fecundity, and fertility in the same treatments during the 2 weeks of observation, and for the biting rate studies described below. Five repetitions were conducted.
Mortality was recorded daily by removing dead individuals. During the two observation weeks, egg collection was initiated 3 days after the first blood meal, and the collection was stopped on the fifth day after the last meal to limit overlap with the second gonotrophic cycle. Paper-lined cups for egg collection were replaced every 3 days to avoid uncontrolled egg hatching. Eggs were maintained under wet conditions for 3 days, allowed to dry, and counted to determine the fecundity rate. Egg fertility was assessed by counting the hatched eggs after immersion in a nutrient broth [40].
Engorgement rate in irradiated Ae. albopictus and Ae. aegypti females under small enclosures
Starting from the fifth day after emergence and continuing for 5 days, irradiated SANG and ARwP females were offered a daily blood meal to monitor their feeding behavior. To allow for the study of a second gonotrophic cycle, this same procedure was also conducted from the 12th to the 16th day. The number of engorged females was tallied within 20 min of placement of the blood meal in the cage. Results obtained with the two populations of females treated at the three radiation doses were compared with those observed using control untreated females. Each of these eight treatments was repeated five times.
Although the present study was focused on Ae. albopictus, Ae. aegypti females irradiated at 50 Gy were similarly tested (in triplicate) in comparison with untreated controls to study the phenomenon in a species that is also a target of SIT and SIT/IIT programs but is not infected by Wolbachia in nature. The hatch rate of irradiated Ae. aegypti was also measured and compared to that of control females to identify a successful radiation treatment.
Host-seeking ability and biting rate of irradiated Ae. albopictus females in large enclosures
The host-seeking behavior of ARwP and SANG females irradiated at 45 Gy was studied in large enclosures and compared with that of untreated individuals based on methods described in a previous study [41]. The trials were conducted outdoor in two large cellular polycarbonate experimental units (LEU, 8.5 × 5 × 5 m L:W:H) with two large lateral openings (L = 8 m; H = 1 m) protected by a mosquito net to promote ventilation and ensure climatic conditions closer to the external environment. The latter were constantly monitored by a CR-10 data logger (Campbell Scientific, Logan, UT, USA), which registered a mean temperature of 34.0 ± 1.0 °C and RH averaging 50.0% ± 5.0%. LEUs contained benches with wet soil and potted plants, which provided refuge for the females and higher local levels of humidity (averaging 61.0% ± 5.0%).
For each experiment, an experimenter (the host) acted as a source of blood, and a second experimenter (the collector) released, recovered, and counted the Ae. albopictus females after they had fed on the host. The host wore a long-sleeved shirt and short pants, exposing only the lower legs to limit the area for the mosquitoes to land on, whereas the collector wore a white tracksuit and white shoes. Both researchers wore mosquito net hats. Four groups, differing in their infection type (SANG or ARwP) and treatment (irradiated and non-irradiated individuals), were compared. Each group consisted of 30 starved females aged 6 ± 1 days. Females were released at the side of the LEU by removing the cover of the cage. The second experimenter then immediately approached the host on the opposite side of the LEU to collect the females that had started feeding. The proportion of blood-fed females was tallied along with the time taken by each individual to reach the host, at 30-s intervals. Female mosquitoes that landed on the host, but did not bite were excluded from the count. The experiments lasted for a period of 15 min, and for each group, six repetitions were performed, alternating the experimental units and the host. At the end of each experiment, an electric mosquito swatter and a powerful aspirator were used to eliminate any mosquitoes remaining inside the LEU. The experiments were conducted in the late afternoon on sequential days during June 2020. A schematic describing the experiment is provided in the supplemental materials (Additional file 1: Figure S1).
In addition to the methods described above, 6 ± 1-day-old females from each treatment group were engorged in the laboratory and used to study the effect of the radiation treatment on their willingness to seek and bite a host 48 h after the first blood meal. After their release in the LEU, the proportion of feeding females and the time to reach the host were again noted.
Quantitative PCR analysis of Wolbachia titer in irradiated Ae. albopictus females
Considering the importance of Wolbachia in modulating the vector competence of infected mosquitoes [42], a qPCR was performed on the strains of this bacterium present in SANG and ARwP females after irradiation at 45 Gy (a dose known to induce full female sterilization in the species) [9]. Results were compared with those obtained using untreated counterparts. In operational SIT programs, mosquitoes are generally irradiated at the pupal stage (24–48 h); however, it is known that pupal age is one of the critical factors that affect the biological response to the radiation dose [43]. Therefore, the effects of the irradiation were investigated by analyzing DNA extracts of whole bodies of 6 ± 1-day-old females developed from pupae irradiated at three ages (26 ± 2, 36 ± 2, and 46 ± 2 h).
The density of Wolbachia is known to vary with the age of Ae. albopictus females [44]. Therefore, to investigate the effects of radiation on the bacterium in the germline and somatic tissues, the titer of the bacterium was measured in ovaries and bodies lacking ovaries of 6 ± 1- and 13 ± 1-day-old females after they were treated with 45 Gy as pupae aged 36 ± 4 h. Untreated females were used as a control.
DNA was extracted from single individuals using the ZR Tissue & Insect DNA Kit MicroPrep (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. When necessary, females were chilled on ice and dissected in phosphate-buffered saline (PBS) to isolate the ovaries from the other tissues. Here we excluded individuals with ovaries that were not intact after the procedure.
Real-time PCR was performed using the Roche LightCycler 96 Instrument (Roche Molecular Systems, Inc., Rotkreuz, Switzerland). Each reaction was performed in triplicate with a final reaction volume of 20 μl (10 μl of the Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.03 μl of 150 nM each primer, and 2 μl of purified DNA) using the following amplification program: initial activation at 95 °C for 120 s, followed by 40 cycles at 95 °C for 15 s and 60 °C for 40 s. The presence of specific amplification products was verified using dissociation curves [44].
Strain-specific primers were used to amplify the wsp loci [45], namely, the wAlbA-wsp and wAlbB-wsp loci in the case of SANG females, and the wPip-wsp loci in the case of ARwP specimens. Wsp plasmid standards were used to generate a standard curve [44]. In order to normalize qPCR data, the Ae. albopictus actin gene was used as a reference for whole-body extracts and extracts obtained after removal of the ovaries and was amplified with the primer pair actAlbqPCR [46]. Owing to the marked decrease in actin gene copies observed preliminarily in irradiated ovaries (Additional file 2: Figure S2), the normalization of qPCR data related to these organs was performed using total DNA (2 μl of purified DNA per reaction, corresponding to 200–300 ng) as reference [47]. For this purpose, a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA) was used.
FISH analysis of the ovaries in irradiated Ae. albopictus females
Based on the results of qPCR, fluorescent in situ hybridization (FISH) analysis was conducted on the ovaries of 13 ± 1-day-old irradiated and untreated females according to the protocol described by previous authors [48]. Two hundred nanograms of the Wolbachia specific 16S rRNA probe (W2: 5′-CTTCTGTGAGTACCGTCATTATC-3′) was added to the hybridization buffer [43]. Tissues were placed on a slide containing a drop of VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and visualized using the Nikon Eclipse E800 confocal microscope and NIS-Elements 4.0 software (Nikon, Tokyo, Japan). An aposymbiotic population of Ae. albopictus [37] was used as the negative control.
Data analysis
Results were expressed as mean ± SE, and the arcsine square root transformation was applied to analyze proportional data. The Levene test and the Shapiro–Wilk test were performed to assess equality of variances and normality, respectively. Statistical analysis was performed using PASW Statistics software (PASW Statistics for Windows, version 18.0. SPSS Inc., Chicago, IL, USA), with the level of significance set at P < 0.05.
The survival curves of the four different treatments for each Wolbachia infection type were compared using the Kaplan–Meier method and the log-rank (Mantel–Cox) test. The Kruskal–Wallis H-test followed by the Conover-Iman test was used to compare fecundity and egg hatch data between treatments within each infection type.
Repeated-measures analysis of variance (ANOVA) was used to analyze the bite data obtained between treatments over each week. Cages were considered the experimental units, and data are expressed as the mean percentage of engorged females per day, adjusted for cage-specific mortality. If Mauchly’s test indicated a violation of the sphericity assumption, the degrees of freedom were corrected using Huynh–Feldt estimates. Multiple comparisons between treatments were assessed using Tukey’s honestly significant difference (HSD) post hoc test. Additionally, one-way ANOVA was performed to compare the mean number of bites per female per treatment during each week of study.
Data regarding the host-seeking behavior under large enclosures were analyzed by assigning a value to each mosquito based on the median of each catching interval to compute the average time to landing. The proportion of engorged mosquitoes was measured based on those retrieved within the defined 15-min interval. Two-way ANOVA was used to analyze the differences between groups in terms of the proportions of females that had bitten the host and to compare average landing times. The Shapiro–Wilk test was conducted to ascertain that the proportions and average landing times were normally distributed.
One-way ANOVA was used to compare within each Wolbachia strain the qPCR data obtained from Ae. albopictus females treated at the three tested pupal ages or untreated. The effect of female aging on the overall titer of Wolbachia was also analyzed by performing one-way ANOVA with data of each strain of the bacterium. Data regarding ovaries and extra-ovarian tissues of the treated or untreated counterparts were similarly analyzed at the two tested female ages. In the case of rejection of the assumptions of equality of variance and/or normality, the Kruskal–Wallis rank-sum test was performed.