Methodology:

Antarctic krill (Euphausia superba) were sampled during research cruise JR19001 aboard the RRS James Clark Ross. Krill swarms were located using a SIMRAD EK60 multifrequency echo-sounder (38, 70, 120 and 200 kHz) and collected using a Rectangular Midwater Trawl (RMT8) net, deployed in the upper 100 m of the water column. Once on board, krill were separated into different sex-stage categories, adults, juvenilles, and where sufficient numbers allowed, adults were separated into males and females. Krill were stored in buckets of underway seawater (0 - 4hours) in a temperature controlled room at 4°C until analysis.

Reflectance spectra of multiple krill (from each krill sex-stage category) were recorded using artificial illumination in a dark room using an ASD FieldSpec 4 Spectroradiometer. This instrument detects radiation in the range 350-2500 nm with a full width at half maximum (FWHM) of 3 nm in the visible range (350-700 nm). Measurements were recorded in a matt black bucket (12 L), with average reflectance of 3.1% within the visible region of the spectrum. The bare fibre fore-optic of the FieldSpec 4 was used, giving a field of view of about 7cm2 at mid-full bucket depth in water. All measurements were taken under a 70 Watt quartz-tungsten-halogen lamp (ASD Illuminator Reflectance lamp) with no other background light. A skylight blocked approach, adapted from Lee et al. (2019), was used to calculate remote sensing reflectance (Rr). A reference reading was taken at the start and end of each experiment using a 20 % grey Spectralon® panel (Labsphere, Inc), which had been calibrated against a reference Spectralon® panel, applying the 8°/T method used by the NERC Field Spectroscopy Facility. Optimal integration time was also set against this 20% panel, giving an in-water signal to noise ratio of between 15 and 170 across the visible region. The spectroradiometer's entrance optics were secured to a tripod and measurements made at nadir (pointing vertically down to the centre of the bucket). The fibre optic end was positioned just below the surface of the water when measuring the spectra of krill or water, removing the effects of surface glint measurements. Reflectance was recorded for each krill sex-stage category in in situ seawater, both 0.2 µm filtered seawater (FSW) and unfiltered seawater (USW) (water sourced from ship's underway system with intake at a depth of 5 m). Initially, the reflectance of only USW or FSW was measured (Rr, water), following which krill were added sequentially (from 5 to 100 krill) with a reflectance measurement made at each addition (Rr, total). All added krill were alive but many individuals were not actively swimming throughout the bucket. A total of 10 reflectance spectra were collected for each addition of krill, with each of these spectra representing an average of 26 readings from the spectroradiometer. The mean of these 10 profiles was calculated to give the raw reflectance measurement (R). The maximum number of krill added was based on the availability of healthy krill at each station sampled. A total of 10 stations were sampled, and the reflectance of krill separated into adults and juveniles were measured at each station. For some of these stations, sufficient numbers made it possible to separate adults further into males and females, and run separate experiments on these categories.

Remote sensing reflectance (Rrs) was calculated for each wavelength (lambda) using the following equation from Lee et al. (2019):

Rrs(lambda) = (R(lambda)/pi*E(lambda))*k

where

E is the reference reflectance from the 20% Spectralon panel (mean of start and end measurements

R is the raw reflectance measurement

k is the Spectralon panel's calibrated reflectance, which, along with pi, converts E from reflected radiance to irradiance

Data processing were carried out using Spectragryph v1.2.

Data collection:

SIMRAD EK60 multifrequency echo-sounder (38, 70, 120 and 200 kHz)

British Antarctic Survey Rectangular Midwater Trawl 8

ASD FieldSpec 4 Spectroradiometer

Spectragryph 1.2

Data quality:

Reflectance measurements have been referenced to a 20 % grey Spectralon® panel (Labsphere, Inc), which had been calibrated against a reference Spectralon® panel, applying the 8°/T method used by the NERC Field Spectroscopy Facility. Data are given here for the full recorded wavelengths from 351-2450 nm. There is high variability in reflectance data < 400 nm due to the lack of light in the UV range from the light source, and so there tends to be a very low-signal to noise ratio in the region below 400 nm, and we advise caution when using data in this region of the spectrum.