Dataset: Goby Histological Inventory

Methodology:

Fish Collection and Rearing (for all histological and SEM analyses) - Mated pairs of E. lori and of E. colini were collected from reef habitats near Carrie Bow Caye, Belize and housed in 75-l aquaria in a flow-through seawater lab at the International Zoological Expeditions (IZE) field station on South Water Caye, Belize, or in a recirculating seawater system at Boston University, USA. Ontogenetic series of E. lori and E. colini larvae were reared in 76-l cylindrical black bins and fed a variety of cultured and wild-caught zooplankton (Figure 1). A detailed description of brood stock maintenance and larval rearing methods can be found in Majoris et al. (2018). Additional post-settlement E. lori and E. colini (settlers) were collected from reef habitats within the South Water Caye Marine Reserve. Field research in Belize and the export of samples from Belize was carried out with the approval of the Belize Fisheries Department. Fish were immersed in cold seawater (2-4 degrees C) for two minutes and then fixed in cold (2-4 degrees C) 10% formalin in seawater (or in phosphate-buffered saline, PBS) for at least two minutes for anatomical study, which is consistent with AVMA Guidelines on Euthanasia of small warm-water fish. Care was taken to ensure that fish did not contact ice directly. Chemical anaesthetic was not used for several reasons, in particular because fixation must occur prior to death to avoid post-mortem changes at the cellular level, and ensure quality of the histological data and specimens prepared for SEM.

Paraffin Histology (taste bud, nose, ear, lateral line and eye analyses) - E. lori settlers wild-caught in Belize in 2011 (n=8, 9-17 mm LS) and larvae reared at Boston University in 2015 in addition to 3 wild-caught settlers (total of 7 fish, 0-30 dph, 3 mm NL - 15 mm SL) were decalcified in Cal-Ex (Thermo Fisher Scientific, Waltham, MA, USA) for 2 hours (6-7.5 mm SL) or 7- 8 hours (>8.5 mm SL) and rinsed in PBS for 2 hours. Then they were dehydrated in ascending series of ethanol and t-butyl alcohol, infiltrated in Paraplast (Thermo Fisher Scientific) for 4 hours under vacuum, and individually embedded. Blocks were sectioned at a thickness of 8 m, serial sections were mounted on slides subbed with 10% albumin in 0.9% NaCl, stained with a modified HBQ stain (Hall, 1986), and coverslipped with Entellan (Electron Microscopy Sciences).

Plastic Histology (taste bud, nose, ear, lateral line and eye analyses) - E. lori raised in Belize in 2015 and 2016 (n=33 0-44 dph, 3 mm NL [notochordal length] - 11 mm SL [standard length]), wild-caught E. lori settlers collected at Belize in 2015 and 2016 (n=5, 9.5-14 mm SL), and E. colini raised at Boston University in 2015 (n=25, 0-50 dph, 3.5-15 mm NL or SL) were prepared for plastic resin histology. Fish larger than 6 mm SL were decalcified in Cal-Ex (Thermo Fisher Scientific, Waltham, MA, USA) for 2 hours (6-7.5 mm SL), 3.5 hours (8-8.5 mm SL), or 7 to 8 hours (>8.5 mm SL) and rinsed in PBS for 2 hours. Fish were dehydrated in an ascending ethanol series (to 95% ethanol), infiltrated overnight in glycol methacrylate resin (Technovit 7100, Electron Microscopy Sciences), and embedded in fresh resin to which polymerization agent had been added. Most of the fish were double embedded - individual fish were embedded in small resin blocks, and 6-8 small resin blocks were re-embedded in a single larger block of resin for sectioning in the transverse plane . Sections (5 m, cut using a Leica 4M2265 motorized microtome with tungsten carbide knife) were individually mounted out of distilled water onto clean slides, dried overnight, stained with 0.5% aqueous cresyl violet for 5 minutes, rinsed in running tap water, air-dried overnight, and coverslipped with Entellan (Electron Microscopy Sciences).

Note: for olfactory size, measurements were taken on histological sections at 25 (position 1), 50 (position 2) and 75% (position 3) along the rostro-caudal length of the olfactory epithelium.