Oil, Clove

The ambon damselfish Pomacentrus amboinensis and lemon damsel P. moluccensis are common site attached species of damselfish (family Pomacentridae) found throughout the Indo-Pacific on shallow reef habitats at the interface between the live coral and rubble reef edge (Fig. S1). Both species have a similar larval duration after a demersal egg phase and settle at similar sizes (P. amboinensis 17.8 d, 11.2 mm SL; P. moluccensis 19.4 d, 10.7 mm SL; [17] ). Metamorphosis is concomitant with settlement and in these species involves a major change in pigmentation (transparent to coloured) that occurs within hours, but involves little obvious change in shape [36] . However, settlement does involve major changes in physiology [37] and it is likely that marked changes also occur in the sensory systems [38] . A laboratory-based habitat selection experiment has previously shown that both species preferentially settle to healthy live coral [39] . Both species settle naturally to patches of mixed live and dead coral. Both are also planktivores as juveniles and eat a similar array of prey items (Text S1). A tagging study of 295 newly settled P. amboinensis on the continuous reef edge found that fish moved little over the first 3 months after settlement (mean = 0.63 m [40] ). It is likely that P. moluccensis has a similar degree of site attachment (pers. obs.).
Research on newly settled P. amboinensis has shown that fish enter the reef with high variability in their behavioural traits (e.g. boldness, aggression) and these traits are displayed in a manner that is consistent on small time scales of hours to days ([41] (link), [42] (link), Mero, Meekan and McCormick unpublished data]. Establishment of dominance hierarchies occurs within minutes of settlement within the species, which can rapidly lead to the eviction of subordinates from small habitat patches [31] (link). Because of the rapid establishment of territories and the high juvenile mortality, it was decided that 60 min was an ecologically relevant time to use for the establishment of residents in the priority experiments for the present study.
The present datasets were collected at Lizard Island (14° 40′S 145° 28′ E) on the northern Great Barrier Reef, Australia, between October 2007 and March 2010. Both newly metamorphosed juveniles and recently settled juveniles from the reef were used for field experiments. Light traps (see [43] for design; small trap) were used to collect both fish species at the end of their larval phase prior to their settlement to the reef. These newly metamorphosed fish were separated by species and placed into 60 L aquaria with aerated flowing seawater. Fish were kept for 24 h and fed newly hatched Artemia sp. twice per day ad libitum to allow recovery from (or acclimation to) the stress of capture, prior to use in experiments. Juveniles were collected from a shallow fringing reef at the back of Lizard Island using a solution of dilute clove oil and hand nets. All fishes used in the experiments were placed into a small clip-seal bag with a small amount of aerated seawater and measured with calipers (±0.1 mm) and then transferred into individually labeled 1 L clip-seal bags for transport. To reduce transport and handling stress, fish in bags were transported to the field site in a 30 L bin of seawater (to reduce temperature fluctuations) under subdued light conditions.

For transcriptional analysis, EHEC was inoculated into 25 ml of LB broth in 250 ml shake flasks at a starting OD₆₀₀ of 0.05, and then cultured at 37 °C for 3 h with agitation (250 rpm) in the presence or absence of clove oil or eugenol (0.005%) for another 2 hours. RNase inhibitor (RNAlater, Ambion, TX, USA) was added to prevent RNA degradation. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA).
qRT-PCR was used to investigate the transcription levels of curli genes (csgA, csgB, csgD, csgF and csgG), type I fimbriae genes (fimA, fimC, fimD, and fimH), other fimbriae genes (ecpA, ecpR, and Z2200), cellulose gene (bcsA), motility genes (flhD, fliA, motB, and qseB), AI-2 quorum sensing genes (luxS and luxR), indole-synthesis gene (tnaA), shiga-like toxin genes (stx1 and stx2), and LEE-encoded regulator genes (ler, espD, escJ, escR, and tir) in EHEC treated with or without clove oil or eugenol (0.005%). Gene specific primers were used and rrsG was used as a housekeeping control (Supplementary Table S3). The qRT-PCR method used was an adaptation of a previously described method30 (link). qRT-PCR was performed using a SYBR Green master mix (Applied Biosystems, Foster City, USA) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures.

Blood samples were collected from five male Labeo rohita (henceforth referred to as Rohu-1 through Rohu-5) from a fish farm located in the District of Rangpur, Bangladesh. The fish were handled as per guidelines of the Ethics Standard Review Committee of Bangladesh Agricultural University (BAU) involving fish and animals (approval no. BAURES/ESRC/2019/Fish/01). Each fish was euthanized using clove oil, dissected, and blood was collected from the heart using a syringe. Each blood sample was placed in an ethylenediaminetetraacetic acid containing vial, and vials were shipped in an insulated container to Mississippi State University for DNA extraction.
High-molecular-weight (HMW) genomic DNA for whole genome sequencing was extracted from 150 µl of blood from Rohu-1 using CTAB lysis buffer followed by the phenol/chloroform purification procedure (Doyle and Doyle 1987 ). The concentration and purity of extracted genomic DNA samples were measured by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of genomic DNA was validated by electrophoresis on a 0.8% w/v agarose gel.
The genomic DNA from Rohu-1 was used to prepare 10 Oxford Nanopore R9.4 MinION flow cells. For each flow cell, 2 to 2.5 µg of genomic DNA and a Nanopore Genomic DNA Ligation Sequencing Kit SQK-LSK 109 (Oxford Nanopore Technologies, Oxford, UK) were used to create a DNA library. For each of the 10 libraries, 700–750 ng of DNA was loaded onto a Nanopore Flow Cell R9.4.1 (Oxford Nanopore Technologies, Oxford, UK) and sequenced on a GridION sequencer (Oxford Nanopore Technologies, Oxford, UK) for 48 h.
Rohu-1 genomic DNA was also sequenced on an Illumina HiSeq X-Ten (2 × 150 bp). In brief, 2 µg of Rohu-1 genomic DNA was used with an Illumina TruSeq DNA PCR-free Library Prep Kit (Illumina, San Diego, CA, USA) to create an Illumina sequencing library. The final DNA-Seq library, which had an insert size range of 350–450 bp, was submitted to Novogene (www.en.novogene.com) for two lanes of PE150 on an Illumina HiSeq X-Ten (Illumina, San Diego, CA, USA) sequencer.
A Hi-C library also was prepared using 100 µl of Rohu-1 blood with the Proximo Hi-C Animal Kit (Phase Genomics, Seattle, WA, USA). The final Hi-C DNA-Seq library was submitted to Novogene (www.en.novogene.com) for one lane of PE150 Illumina HiSeq X-Ten (Illumina, San Diego, CA, USA) sequencing.
Lastly, Rohu-1 blood cells were embedded in agarose and HMW DNA was isolated according to the Bionano Prep Frozen Blood Protocol (Bionano Genomics, San Diego, CA) . The extracted DNA molecules were labeled with the Direct Label and Stain (DLS) DNA Labeling kit (Bionano Genomics, San Diego, CA). Once labeled and stained, the DNA was imaged on the Bionano Saphyr instrument (Bionano Genomics, San Diego, CA).

Animal procedures were performed in accordance with Czech legislation (section 29 of Act No.246/1992 Coll. on Protection of animals against cruelty, as amended by Act No. 77/2004 Coll.). Animal handling complied with the relevant European guidelines on animal welfare (Directive 2010/63/EU on the protection of animals used for scientific purposes) and the recommendations of the Federation of Laboratory Animal Science Associations. The animal experiments have been approved by the Ministry of Education, approval ID: MSMT-18301/2018-2.
Throughout the month of August 2020, we studied rainbow trout O. mykiss (mean body mass of 120 g) reared in a commercial inland fish farm, in the South Bohemian region of the Czech Republic. Fish displaying visible signs of PKD such as pale gills, abdominal distension, and lethargy were transported alive in oxygenated water/bags to the Institute of Parasitology of the Biology Centre of the Czech Academy of Sciences (České Budějovice, Czech Republic). These fish were compared to an additional 15 naïve SPF fish (mean body mass of 30 g), reared in experimental recirculating systems of the Faculty of Fisheries and Protection of Waters, University of South Bohemia in the Czech Republic. Data from these fish are referred to as coming from “control” fish in the text.
Upon arrival, we euthanized fish with clove oil and bled them from caudal veins using heparinized syringes and needles. Upon dissection, we prepared imprints from their swollen kidneys and stained them according to the manufacturer’s instructions with the Kwik–Diff Kit (Richard Allen Scientific, San Diego, CA, USA), consisting of methanol fixation followed by eosin and methylene blue staining. Light microscopy was used to identify T. bryosalmonae parasites. All fish in the infected cohort had kidney swelling indices between 3-4 according to the scale and parameters established by Clifton-Hadley et al. (20 (link)) and variable signs of spleen swelling, fibrosis, and of gill and liver pallor.