Smz745

Fish measurements were made seven times, at four-week intervals, over the course of both experiments (t₀, t₁, t₂, t₃, t₄, t₅, t₆). To measure them, fish were removed from their tank and placed in a small cylinder filled with tank water for transport. They were then picked up from the cylinder using a plastic spoon and briefly placed on a microscope slide with an engraved 1 mm gridline system as a scale. The fish were photographed laterally using a stereoscopic microscope (Nikon SMZ745: 7.5 × zoom and 115 mm working distance) with a camera (Canon EOS60D). ImageJ was used to determine the standard length (SL; length measured from the distance between the tip of the snout to the last vertebrae) to 0.01 mm from each photograph three times. The average of the three measurements for SL were used as the monthly measurement for each individual. After being placed back in their cylinders, all fish swam directly to their anemones. Fish growth per month was calculated as the difference in size between two time points.

Wild-caught specimens of stable flies were used in this study, which followed the WHO susceptibility test guidelines [28 ]. To reduce the factors related to the physiological status of specimens, only nonblood-fed specimens were used for testing. The specimens were collected from a horse farm in Nakhon Pathom Province, Central Thailand (13°45′43.4″ N 100°08′15.7″ E), between March and May 2021. This farm did not use insecticides. The Nzi traps [57 (link)] were placed at the collection site from 16:00 to 18:00. The collected flies were stored in plastic cups and then transported within Styrofoam boxes containing ice packs to the Pharmacology Laboratory of the Faculty of Veterinary Science, Mahidol University. Both male and female stable flies were used for insecticide testing. In particular, the nonblood-fed specimens with undamaged physical characteristics (i.e., antenna, wing, and leg) were selected from all collected specimens under a stereomicroscope (SMZ745, Nikon, Tokyo, Japan) without anesthesia. In the laboratory, these specimens were maintained at 27–29 °C and 70–80% relative humidity until they were used for testing (within 1–2 h).

The assessment of female fecundity after RNAi treatment (dsburs, dspburs, dsrk and dsGFP) involved the examination of ovarian development, as well as the quantification of egg number, size and hatch rate. To observe ovarian development, the ovaries were dissected from the female whiteflies and placed in pre-cooled PBS following a 5-day RNAi treatment. Imaged were captured using a stereomicroscope (Nikon SMZ745, Japan). For fecundity determination, 10 female whiteflies were used for each treatment group and the total number of eggs laid on the leaflet per female was counted over a span of 7 days. The experiment was repeated three times. The egg hatching rate was calculated as the percentage of eggs that successfully hatched compared to the total number of eggs laid. Egg size was measured and imaged using the stereomicroscope.

The entire worm population on each plate was collected in 1 ml of M9 medium. This initial stock was diluted 1:10 in M9 medium to count the total population or in 1% SDS to count the amount of dauers after 20 to 30 min of incubation (13 (link)). Ten microliters of each dilution was used to count the total population and the dauer larvae, respectively, under a Nikon SMZ745 stereomicroscope. Each condition was scored 3 times, and the amounts of dauers were plotted as percentages of the total populations of animals.

Cohorts of ≤50 midges were fed EHDV-2 infected blood using a Hemotek membrane feeding system (Hemotek Ltd., Blackburn, UK) following published protocols [33 (link)]. Parafilm was used as the membrane for blood feeding using defibrinated bovine blood (Hemostat laboratories, Dixon, CA, USA) in three treatments: Can-Alberta strain, Florida strain, and control (blood without the addition of virus). Blood was collected before and after each blood feeding trial to measure viral titer and to ensure titers were similar between the two infected groups. The calculated titer of virus in the two virus fed treatments were each 5.5 log₁₀ plaque forming unit equivalents (PFUe)/mL. Midges were allowed to blood feed for one hour after which blood-fed females were sorted from the males and unfed females using a CO₂ pad (LabScientific Carbon Dioxide Staging Platform, Cat #BGSU-12, Highlands, NJ, USA) and a stereoscopic light microscope (Nikon SMZ 745). Blood-engorged females were sorted into cups of ≤25 individuals, separated by treatment. After blood-feeding, midges were provided 10% sucrose solution ad libitum on cotton pads and maintained at 25 ± 1 °C, 60–80% RH, and 12:12 (L:D) cycle until processing.

Eggs were collected on juice agar plates in 30 min intervals and immediately exposed to one session of heat shock at 37°C for 30 min or kept as controls. This selection was random. Embryos were thereafter kept in a climate‐controlled 22°C incubator for approximately 2 h, dechorionated in 3.5% bleach, and staged under SMZ 745 (Nikon) bright‐field microscope using the criteria for Bownes' stage 5 (Bownes, 1975 ), including formed cells at egg surface and round pole cells at the posterior axis. Single embryos were collected in 2 μl RNase‐free water with an RNase inhibitor and ruptured with an RNase‐free needle. One 5 mm ∅ metal bead (Qiagen) and 500 μl Qiazol (Qiagen) were added per sample and the samples were homogenized for 2 min at 40 Hz using TissueLyser LT (Qiagen). n = 24 single embryos per condition.