BioSample

We collected over 400 Ae. albopictus larvae and pupae from approximately 20 tires located at a used tire yard in Manassas, VA, in 2008. This strain was reared in the laboratory on a non-diapause inducing (NDI) long-day photoperiod (16 h light, 8 h dark) at 21oC and ca. 80% relative humidity for ten generations as described in Armbruster and Hutchinson (2002) and Armbruster and Conn (2006). Approximately 500 male and female pupae were placed into each of six cages, three of which were maintained under an NDI photoperiod, whereas the others were maintained under a diapause-inducing (DI) unambiguous short-day photoperiod (8 h light, 16 h dark). Females were bloodfed to repletion 10-13 days after eclosion on a human host. We collected eggs from 6-hour oviposition periods over the course of 3 days. On each day, we placed a small brown jar half-filled with ca. 50 ml of dI water and lined with unbleached seed germination paper into each cage 4 hours after “dawn”, and removed the jar after 6 hours. Egg papers reserved for the 135-141 h developmental stage and for diapause incidence measurements were slowly dried ca. 48 h after oviposition. RNA extraction from eggs reserved for the 72-78 h development period commenced 72-73 hours after egg papers were removed from their cages. Eggs were placed directly into TRI® Reagent (Sigma Aldrich, St. Louis, MO). Eggs at 135-141 h of development were brushed from egg papers using camel’s hair brushes (Thermo Fisher Scientific, Hampton, NH) 135-136 hours after removal from the cage. Eggs were ground in TRI® Reagent in 2 ml glass Tebroek tissue grinders (Corning, Corning, NY), followed by total RNA extraction and isopropanol precipitation, according to manufacturer’s instructions.