Fig 2: EAB neonate larvae. (A) EAB larva hatching from the egg; (B) Neonate larva size compared to egg; (C) neonate larva (~0.2 cm) on a scale of 1 cm. Olympus SZ61 Stereo Zoom Microscopy was used to amplify the EAB larvae and eggs and the pictures were taken by Olympus DP12 Microscope Digital Camera and iPhone 6S.

Fig 3: Fetal development at 16 days of gestation. Fetuses length (a) and weight (b), and fetal: placental weight ratio (c) of young and senile groups. We considered the average of the litters per male, 14 litters as experimental unit (5 litters in the senile group and 9 litters in the young group). The photos were performed with a megapixel digital color camera (Olympus LC30, Olympus®, Munster, German) attached to a stereomicroscope (Olympus SZ61, Olympus®, Tokyo, Japan) at ×8 magnification. Different superscript letters in each bar represent p < 0.05, as indicated by statistical T-test.

Fig 4: Rhipicephalus sanguineus feeding tissue contraction assay. (A) Unfed female R. sanguineus ticks (2- to 3-month-old) were dissected under tick physiological saline to expose the pharynx-esophagus. Tick endogenous pyrokinin (B) and PK analog (C) increase the contractions of the pharynx-esophagus: After dissection, ticks were transferred to a Sylgard plate into fresh saline (100 µl). After 5 min of adaptation to the environment, the solution was replaced with 100 µl of saline, and tissues were filmed to register any background contractions. The solution was replaced by 100 µl containing 10 µM of a scrambled peptide of PK (RNFSRINTPa) as negative control and incubated for 3 min before the tissue response was filmed. Tissue was then rinsed (5 × 100 µl of saline), and the saline was replaced with 100 µl of tick endogenous pyrokinin (Rhisa-CAPA-PK1: RSNTFTPRIa), or a PK analog (1944-P8: MS[PEG8]-YFTPRLa), both at 10 µM. After 3 min of incubation, the tissue was filmed. Tissue responses to all treatments were filmed for a continuous 1 min using a Lumenera Infinity-1 color camera (Teledyne Lumenera, Ottawa, ON, Canada) installed on an Olympus SZ61 trinocular stereo microscope (Olympus, Center Valley, PA, United States), and contractions in the video were counted by the same operator (mean ± SEM). A repeated measures one-way ANOVA analysis (P < 0.05) followed by a Tukey’s multiple comparisons test were performed to determine the difference in response to different treatments on the same tick (N = 6–7 ticks); different letters in the figure indicate significant difference, P < 0.05.

Fig 5: Mosquito (female) of the Culex pipiens complex. Photograph with a digital camera SC30 (OLYMPUS, Tokyo, Japan) under a stereomicroscope OLYMPUS SZ61 (12× magnification).