The Genetic Basis for Salivary Gland Barriers to Arboviral Transmission

Abstract

Arthropod-borne viruses (arboviruses) infect mosquito salivary glands and then escape to saliva prior to virus transmission. Arbovirus transmission from mosquitoes can be modulated by salivary gland infection barriers (SGIBs) and salivary gland escape barriers (SGEBs). We determined the influence of SGIBs and SGEBs by estimating the quantitative genetic contributions of Aedes aegypti half-sib families (Mapastepec, Mexico) infected with three dengue 2 (DENV2), two chikungunya (CHIKV), and two Zika (ZIKV) genotypes. We determined virus titer per salivary gland and saliva at seven days post-infection and virus prevalence in the half-sib population. CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB. However, virus titer and prevalence due to additive genetic factors in the half-sib family displayed a significant narrow-sense heritability (h²) for SGIB in two of the three DENV2 genotypes and one CHIKV and one ZIKV genotype. SGEBs were detected in all seven virus strains: 60-88% of DENV2 and 48-62% of CHIKV or ZIKV genotype infections. SGEB h² was significant for all CHIKV or ZIKV genotypes but not for any of the DENV2 genotypes. SGIBs and SGEBs exhibited classical gene-by-gene interaction dynamics and are influenced by genetic factors in the mosquito and the virus.

Keywords: Aedes aegypti; arbovirus; quantitative genetics; salivary gland escape barriers; salivary gland infection barriers.

Figure 1

Structure of Aedes spp. salivary glands [3]. (A) Schematic representation of salivary glands of a mosquito at 2 days post-emergence. (B) Transverse section through a median lobe of the salivary gland showing the acinar cells and their relationship to the salivary duct. (C) An enlargement of B showing a cartoon of a transverse-section of salivary gland indicating mature virus penetrating the basal lamina to establish an infection in the acinar cells (A,C) and then penetrating the plasma membrane to fill the apical cavity and/or accumulate in the acinar cell junctions. Pink arrows trace arbovirus entry and movement in individual acinar cells leading to the virus’ eventual release through the apical cavity (yellow lines). Salivary gland escape (SGE)1 and SGE2 refer to potential barriers (entry into the apical cavity and acinar cell junctions, respectively) for virus escape from salivary glands (D) Immunofluorescence of salivary gland lobe infected with DENV-2 Merida (Cosmopolitan) at 7 dpi shows that mature viral particles clearly accumulated in acinar cell junctions (red arrow). (E) Uninfected salivary glands showed no immunofluorescence signal. N: nuclei. SD: salivary duct. AC: apical cavity for saliva storage. BL: basal lamina. Cyt: Cytoplasm. CM: Cell membrane. DLL: distal lateral lobe, ML: medial lobe. * Represents a virus cluster (1C).

Figure 2

Individual capillaries observed under the microscope for the presence of saliva and assigned saliva categories based on total volume. The volume of saliva was calculated using the cylinder volume formula (V = π (r² × h) = 3.1416 × 0.01 × h = mm³ or µL) and measuring the height of the saliva with a digital fractional caliper (precision: ±0.02 mm), as described previously.

Figure 3

Method for generating nested half-sib design used for calculating estimates of genetic and environmental variances and covariances.

Figure 4

Titer of Mapastepec Aedes aegypti mosquitoes 7 days after intrathoracically injecting with 500 plaque forming units (PFU) of virus. Titers were determined by plaque assay in the whole mosquito for each viral strain. Means and 95% confidence intervals are shown. * = p < 0.0001.

Figure 5

(A) Average numbers of log₁₀ (PFU/mL) per salivary gland for all seven viral genotypes. (B) Average numbers of log₁₀ (PFU/mL) per saliva were calculated for all seven viral genotypes. Horizontal lines over pairs of genotypes in either graph indicate that average log₁₀ (PFU/mL) per salivary gland did not differ significantly.

Figure 6

Distribution of phenotypes log₁₀ (PFU/mL) per salivary gland in half-sib families for the seven virus strains. Each column of points arising from the x-axis represents a sire family. In each column, the distribution of individual salivary glands titers arising from that sire appear as gray circles. Mean sire values and their 95% confidence intervals are displayed in red. The mean of dam families in each sire appear in green. (A) DENV2 American genotype, (B) DENV2 Asian genotype, (C) DENV2 Cosmopolitan genotype, (D) CHIKV ESCA-IOL genotype, (E) CHIKV Asian genotype, (F) ZIKV Asian genotype, and (G) ZIKV African genotype.

Figure 7

Correlation analysis to test whether the average log₁₀ (PFU/mL) virus in the salivary glands are negatively correlated with the magnitude of heritability.

Figure 8

Distribution of phenotypes log₁₀ (PFU/mL) per saliva in half-sib families for the seven virus strains. Each column of points arising from the X-axis represents a sire family. In each column, the distribution of individual saliva titer arising from that sire appear as gray circles. Mean sire values and their 95% confidence intervals are displayed in red. The mean of dam families in each sire appear in green. (A) DENV2 American genotype, (B) DENV2 Asian genotype, (C) DENV2 Cosmopolitan genotype, (D) CHIKV ESCA-IOL genotype, (E) CHIKV Asian genotype, (F) ZIKV Asian genotype, and (G) ZIKV African genotype.

Figure 9

Correlation between virus titer log₁₀ (PFU/mL) per saliva and volume of saliva for the seven virus strains. Each column of points arising from the X-axis represents a volume of saliva assigned. In each column, the distribution of individual saliva titers appears as black circles, also the number of positive saliva/total number of saliva per volume and prevalence is in parenthesis. Mean titer values and their 95% confidence intervals are displayed in red. Red arrow indicates non-salivated samples (n = 5). Total number of saliva samples tested and p value per virus strain are indicated. (A) DENV2 American genotype, (B) DENV2 Asian genotype, (C) DENV2 Cosmopolitan genotype, (D) CHIKV ESCA-IOL genotype, (E) CHIKV Asian genotype, (F) ZIKV Asian genotype, and (G) ZIKV African genotype.