Ua 002 08

The initial aquaculture water was adjusted to a salinity of 25 PSU by adding fresh water to concentrated seawater (approximately 150 PSU) purchased from a reservoir pond for a salt farm in Samut Songkhram Province, Thailand. During the experiment, the salinity in each pond was allowed to fluctuate naturally with rainfall or evaporation and tested every week using a salinity refractometer (Master-S/Mill Alpha; Atago Co. Ltd., Tokyo, Japan). Water temperature and pH were not controlled throughout the experimental period. Water temperature was measured every hour using a temperature logger (UA-002-08; Onset computer Inc., Massachusetts, USA). pH was measured every morning (9:00) using a pH meter (HI98127; Hanna Instruments Inc., Rhode Island, USA). Alkalinity was measured every 2 weeks using the potentiometric titration method [23 ].
Total ammonia-N and phosphate were analyzed using the Nessler and ascorbic acid methods, respectively, via a portable spectrophotometer (DR 2400; HACH Co. Ltd., Loveland, CO, USA). Nitrite-N and nitrate-N were analyzed using the colorimetric method of the American Public Health Association [23 ].

Five individual P. lobata colonies were selected as source colonies from the nearshore and offshore sites for the reciprocal transplant experiment. All samples were identified as P. lobata through colony morphology, corallite skeletal morphology⁴⁶ , and DNA analysis of the Histone2 (H2) marker (Table S2)^{12 (link)}. Ten small fragments (approximately 1.5 cm in diameter) were collected from the upward facing surface of each source colony on April 15, 2015. One sample was immediately frozen on-shore using liquid nitrogen and another was fixed in 10% Z-fix (aqueous buffered zinc formalin) in filtered seawater for establishing baseline data. Half of the remaining coral fragments from each colony were cross-transplanted to the other location, and the remaining half were back-transplanted to their original location for 30 days (Fig. 1). Temperature and light intensity profiles were measured by deploying a data logger (HOBO, # UA-002-08, Onset Computer) at each site. Extensive chemical and physical data of Maunalua Bay’s sediments and water were available from previous studies^{7 ,9 ,10} . At the end of the experiment, one fragment of each source colony at each location was flash frozen on site using liquid nitrogen and stored at – 80 ℃ at KML for protein analyses. The rest of the fragments (up to three per source colony per site) were fixed in Z-fix for physiological assays.

Diel changes in ambient incident photon irradiance (continuously measured via Odyssey light loggers; Dataflow Systems, Christchurch, NZ), water-column pO₂ (via O₂ micro-optodes; OXF500PT, Pyroscience, Aachen, Germany; connected to a 4-channel Firesting meter, PyroScience, Germany), and water-column temperature (via HOBO temperature data loggers; UA-002-08, Onset Computer Corporation, Bourne, MA, USA) were recorded over ~24 h within the enclosures. All sensors were calibrated according to the manufactures instructions, mounted on a metal spear and positioned at leaf canopy height. Logging (1 Hz) by all data loggers was synchronized with the logging of microsensors used for the intra-tissue measurements.

For studies at temperature below developmental thresholds, H. halys were collected by hand from building structures in Portland, OR metropolitan area. The timing of insect collections (Mid-November until Mid-December) coincided with the beginning of natural diapause for H. halys in this region. Halyomorpha halys adults were kept in groups of 20 inside 25 × 50 × 75 mm cardboard shelters fitted with flat 50 × 75 mm cardboard inserts and exposed to 8 °C conditions with 16:8 D:L photoperiod, simulating the average dormant temperatures in Western Oregon. This temperature is known to be below the lower developmental and reproductive thresholds of H. halys [4 (link)]. Seven shelters were maintained under this rearing regime for three months. To verify that the internal temperature of the shelters was accurate, the probe of a thermocouple data logger (model UA-002-08; Onset, Bourne, MA, USA) was inserted at the base of each shelter and secured with masking tape. We determined mortality of diapausing H. halys in all shelters every 3–4 days. Any individual that did not move when its abdomen was prodded was considered dead.