During navigation, landmark processing is critical either for generatingan allocentric-based cognitive map or in facilitating egocentric-basedstrategies. Increasing evidence from manipulation and single-unitrecording studies has highlighted the role of the entorhinal cortex inprocessing landmarks. In particular, the lateral (LEC) and medial(MEC) sub-regions of the entorhinal cortex have been shown to attendto proximal and distal landmarks, respectively. Recent studies haveidentified a further dissociation in cue processing between the LECand MEC based on spatial frames of reference. Neurons in the LECpreferentially encode egocentric cues while those in the MEC encodeallocentric cues. In this study, we assessed the impact of disruptingthe LEC on landmark-based spatial memory in both egocentric andallocentric reference frames. Animals that received excitotoxiclesions of the LEC were significantly impaired, relative to controls,on both egocentric and allocentric versions of an object–placeassociation task. Notably, LEC lesioned animals performed at chance onthe egocentric version but above chance on the allocentric version.There was no significant difference in performance between the twogroups on an object recognition and spatial T-maze task. Takentogether, these results indicate that the LEC plays a role in featureintegration more broadly and in specifically processing spatialinformation within an egocentric reference frame. Spatial memory and navigation require us to learn and remember the locations oflandmarks within our environment. These landmarks can take numerous formsfrom large geographical features to small objects within our localenvironment. We can use landmarks to form an allocentric map of the externalworld that allows flexible navigation, including the generation ofshortcuts; the cognitive map . Wecan Place cells in the hippocampus fire in consistent locations relative tolandmarks providing a potential neural mechanism to support the cognitivemap . Place cStudies of LEC have shown a clear lack of spatially modulated signals , althougHowever, all of the studies covered so far involve testing the ability to uselandmarks to support allocentric spatial memory. Recent studies havedemonstrated that LEC neurons show clear egocentric coding while MEC signalsare dominated by allocentric cues when rats are foraging in an openenvironment with no local cues . In thisn = 14; averageweight at start of experiment: 359 g). Behavioural testing wasconducted 5 days a week during the light cycle. The maintenance oflaboratory animals and their use in scientific experiments compliedwith national  Act, 1986) andinternational (European Communities Council Directive of 24 November1986 (86/609/EEC)) legislation governing the maintenance of laboratoryanimals and their use in scientific experiments. Local approval wasalso received from the St Andrews Animal Welfare and Ethics Committee.For both experiments, animals had free access to water while in theircages. Specifically for experiment 2, animals were food restricted tono less than 85% of their free feeding bodyweight. This was done tomotivate animals during behavioural testing, which involved a foodreward.Male Lister Hooded rats  were housed in groups offour on a 12-h light/dark cycle (Three recognition memory tasks were conducted: standard objectrecognition (OR) and two versions of object–place (OP) recognitionpromoting an egocentric and allocentric strategy respectively. Allthree tasks were run using two objects placed in a 67 cm square boxwith 40 cm high walls. The box could be set up as two different‘contexts’ by swapping in/out the walls and floor of the box. The‘white’ context had floor and wall inserts painted white. In the‘stripes’ context, the walls and floor inserts were painted with blackand white vertical stripes (5 cm width) with an additionalplastic-coated metal mesh overlaid on the floor. The two objects wereattached to the box floor with Dual Lock Velcro ,side-by-side approximately 15 cm apart and towards the north wall see. ObjectsFor the spatial task, a modified T-maze with 3 cm high walls was set up80 cm from the floor. The maze had a central stem that extended 60 cmto the T-junction before turning off to the left and right arms. Onlya portion of the central stem was used for the experiment see .The mazFollowing 1 week of extensive handling to habituate the rats to theexperimenter, rats were individually habituated to contexts (4 days)and then to novel objects within contexts (4 days). The two contextconfigurations were not relevant to the current hypotheses, and nosignificant effects of context were found on task performance (datanot reported). The use of the two contexts and order of presentationof contexts was counterbalanced within and across tasks for bothgroups of rats. Rats were then tested on a series of recognition tasksin the following order: OR, OP (egocentric) and OP . Eachtask was run for 4 days, with rats receiving a sample and test trialon each of those days. At the end of the sample trial, rats wereplaced in a holding cage for 1 min while the box was cleaned andconfigured for the test trial.In the sample trial, rats were given 3 min to explore two identicalobjects (e.g. toy lamps). The trial ended at the end of 3 min or whenrats explored both objects for 15 s each, whichever was shorter.During the test trial (3-min duration), rats were exposed to a newcopy of the previously seen object (e.g. toy lamp) as well as a novelobject not previously seen (e.g. martini glass). Memory for theobjects from the sample trial would be expected to drive preferentialexploration of the novel object (e.g. martini glass) in the testtrial. Identity and position of the novel object were counterbalancedwithin and across days for the lesion and sham groups. Theexperimenter placed the rat in the box facing the south wall at thestart of both trials see .In the sample trial, rats were exposed to two different objects  for 3 min. During the test trial (3-min duration), twoidentical copies of one of the previously seen objects (e.g. toy cars)were placed in the box for rats to explore. Integrated memory ofobject and location from the sample trial would be expected to drivepreferential exploration of the object in the location in which it hadnot previously been experienced in the test trial. Two versions of theOP task were run, in which either an egocentric or allocentricencoding of space was encouraged. The key difference between theegocentric and allocentric tasks was the direction in which rats wereplaced at the start of the test trial see . For theRats were habituated to the T-maze for 3 days. On the first day, ratswere placed at the base of the central stem and allowed to explore themaze for 5 min. On the second day, a food reward  was placed in both the east and west arms of the maze.Rats were placed in the starting point and allowed to explore the mazeuntil they had found and eaten both rewards or 10 min had elapsed,whichever happened sooner. On the third day, a food reward was onlyplaced in the west arm. Rats were allowed to find and eat the foodreward in the west arm and also explore the non-rewarded east arm.After completing habituation, rats were trained on a spatial task for7 days, receiving four trials per day. Rats were trained to turn lefton to the west arm to receive a food reward see . In evern = 6) and sham (n = 8)groups were initially anaesthetized using isoflurane  in an induction box. They were thenplaced in a stereotaxic frame  whereanaesthesia was maintained via a facemask mounted on the incisor bar. A pre-surgical analgesicRimadyl  wasadministered subcutaneously. After shaving the animal’s scalp, amidline incision was made and holes drilled bilaterally at stereotaxicco-ordinates targeting LEC: −6.5 mm from Bregma; ±4.5 mm from themidline (measured on the skull surface). Dura was cut using the benttip of a 30-gauge needle and the pipette lowered into the brain at a10° angle to 6.4 mm below dura. For animals in the lesion group,188 nl of ibotenate  was infused by pressure ejection from a drawn glassmicropipette (tip diameter 30–40 microns) and left in situ for 5 minafter infusion. Sham controls underwent the identical procedurereceiving only the vehicle solution (sterile phosphate buffer). Ratswere given 7 days to recover from surgery before behavioural testingbegan.Group sizes were determined based on previous studies showing robusteffect sizes for rats performing OR and OP tasks in our laboratory. Rats in both thelesion and transcardially perfused with phosphate-buffered saline (0.9%).This was followed by at least 250 ml of paraformaldehyde solution (4%made up in 0.1% phosphate buffer solution). Brains were then extractedand placed overnight in 20% sucrose solution (made up in 0.1%phosphate buffer).Brains were immersed in egg yolk within 24-well tissue culture platescontaining paraformaldehyde (40%) in the empty neighbouring wells.These were left for 5 days to allow the egg to fix onto the outside ofthe brains. Brains were subsequently cut into 50 µm coronal sectionson a freezing microtome and then mounted 1:4 sections onto slides.Sections were then stained on the slides with cresyl violet. To dothis, slides were placed in a slide holder and then submerged in glassvases of xylene (2 min), 100% alcohol (1 min), 50% alcohol (1 min),water (1 min), cresyl violet (2 min), running water (5 min), 50%alcohol (1 min), 100% alcohol (1 min) and finally cleared in xylene.Slides were individually removed from xylene and coverslipped usingDPX mountant .Slides were viewed under a light microscope (Leitz Diaplan) atmagnification ×10 and ×4. The extent of lesioned area was judged bythe lack of cell bodies or by cells that were shrunken and damaged.Lesion damage was drawn onto 10 standardised sections of LEC withreference to For the three recognition memory tasks, animals were scored to beactively exploring an object when their noses were within 2 cm of theobject. The exploration times for the two objects were then convertedinto discrimination ratios (discrimination ratio = (time at novelobject – time at familiar object) / (time at novel object + time atfamiliar object)) to determine an animal’s relative exploration of thenovel versus familiar object or OP association. The discriminationratio calculated here is equivalent to the D2 measure used by For each trial on the spatial T-maze task, animals were judged to havemade a choice when all four of their paws were simultaneously beyondthe entrance to either the left or right arms. Animals taking a leftturn were judged to have made a correct choice while those turningright were classified as making an incorrect choice. Responselatencies were also measured on each trial by recording how long (inseconds) it took animals to make a correct or incorrect choice fromthe time they were placed at the base of the central stem.P value, 5000 reshuffles of the controland LEC groups were performed. The P value reportedis the likelihood of observing the effect size, if the null hypothesisof zero difference is true. www.estimationstats.com; t tests were also used to assess whetherthe average discrimination ratios for the lesion and sham groups weredifferent to chance (0) on the various recognition memory tasks.Additional paired-samples t tests were conducted forboth groups to compare discrimination ratios between the first andsecond halves of the OP allocentric task. In experiment 2, 2 × 7 mixedANOVAs, with lesion group  as the independent factor andtraining day (days 1–7) as the repeated measures factor, wereperformed for both accuracy and latency during the spatial T-mazetask. One-sample t tests were conducted to assesswhether accuracy performance for the lesion and sham groups wasdifferent to chance (0.5) for each test day. Bonferroni correctionswere applied to post hoc comparisons conducted on significant main andinteraction effects. A Greenhouse–Geisser correction was applied ininstances where the sphericity assumption was violated for therepeated measures factor (training day). Statistical analyses wereperformed using IBM SPSS Statistics 25.0®.In experiment 1, separate univariate analyses of variance (ANOVAs) wereconducted on the discrimination ratios and exploration rates in thetest phase and sample phase for each of the three recognition memorytasks. To determine the likelihood of the reported effects persistingacross larger samples, we ran data analysis with bootstrap-coupledestimation . A totalHistological analysis determined that five of six rats in the lesiongroup had successful bilateral lesions of the LEC. Lesion damage fromrats with the largest and smallest lesions is depicted in F = 2.82,p = .061, t(7) = 8.76,p < .001) and lesion(t(4) = 8.93,p < .001) groups had discrimination ratiossignificantly greater than chance. Thus, rats in both sham and lesiongroups were able to remember objects. There was no significantdifference in the total amount of time exploring objects in general(time spent at novel + familiar objects) between rats in sham and LEClesion groups  = 1.19,p = .149, Analysis of performance on the object recognition (OR) task replicatedour previously reported effects , 2013b.F = 5.09,p = .023, t(7) = 3.82,p = .004), demonstrating that they preferredexploring novel OP associations and therefore had remembered familiarOP associations. In contrast, rats in the LEC lesion group showed nosuch preference for exploring novel OP associations(t(4) = –0.81,p = .470), showing chance-level performance. Therewas no significant difference in the total amount of time exploringobjects between rats in sham and LEC lesion groups in either thesample  = 1.24,p = .289, F = 2.37,p = .076, As both sham and lesioned rats were able to demonstrate memory forfamiliar objects, we could now assess their performance on tasks thatrequired them to remember the association of objects and the locationsin which they were experienced. Analysis of performance on the OP(egocentric) recognition task also revealed effects that replicatedthose we have previously reported .AverageF = 12.23,p = .003, t(4) = 2.76,p = .025), as were rats with sham lesions(t(7) = 14.43,p < .001). Thus, both groups recognised familiarOP associations in the test phase. There was no significant differencein the total amount of time exploring objects between rats in sham andLEC lesion groups in either the sample = 0.03,p = .870, F = 1.13,p = .156, Similar to the egocentric version of this task, average discriminationratios were significantly worse for LEC lesioned animals relative toshams  = –3.47,p = .013) suggesting that these animals were onlyable to recognise the position of objects within an allocentricframework after some time in the environment. This was supported byone-sample t tests, which revealed that the LEClesion group was performing at chance in the first half of sessions(t(4) = 1.95,p = .062) but above chance during the second half ofsessions (t(4) = 3.90,p = .009). Sham-lesioned animals did not havesignificantly different discrimination ratios between the two testinghalves (t(7) = 1.71,p = .066). Interestingly, one-samplet tests revealed that sham animals performedabove chance in the first half of sessions(t(7) = 6.23,p < .001) but at chance in the latter half(t(7) = 0.64,p = .271). As would be expected, this suggests thatnormal animals quickly recognise the position of objects within anallocentric framework and explore the novel configuration in the earlystages of the trial with discrimination becoming less prominentthrough the course of the trial as the novel OP configuration becomesmore familiar.To examine the deficit seen in the LEC lesion group in more detail, wecompared discrimination ratios between the first and second halves ofthe test sessions . One post(7) = 3.87,p = .003) and LEC lesion groups(t(4) = 10.86,p < .001) spent considerably more time exploringobjects in first half relative to the second half of test sessions = 23.23,p = .001, F = 3.02,p = .011, F = 2.39,p = .037, F = 22.85,p = .001, F = 5.03,p = .047, F = 6.92,p < .001, F = 0.57,p = .638, t tests furtherhighlighted a difference in accuracy between the two groups on thefirst 2 days of training. LEC lesioned animals showed above-chanceaccuracy (day 1: t(4) = 6.53,p = .003; day 2:t(4) = 3.50,p = .025) while animals in the sham group remained atchance level performance (day 1:t(7) = 1.53, p = .170;day 2: t(7) = 1.43,p = .197). Both groups showed above-chance accuracyon the last 5 days of training.Given that rats were impaired at both types of OP recognition task, wewent on to examine whether LEC lesions produced a deficit in astandard test of spatial reference memory on a T-maze. Critically,this task does not require rats to integrate items with spatiallocations and rather is a non-associative test of spatial memory. Ourprevious studies have demonstrated that LEC is necessary for theassociation of features of an event suggesting that non-associativespatial memory should not be affected. LEC animals were not impairedin learning this simple spatial task. Interestingly, the rats with LEClesions were more accurate than the sham animals at the beginning oftraining. This was confirmed with a 2 × 7 mixed ANOVA that revealedsignificant main effects of group = 4.10,p = .001, F = 4.57,p = .054, F = 1.78,p = .116, t tests highlighted adifference in accuracy between the two groups during the initial daysof training (see t(5) = 2.45,p = .058, shams:t(7) = 1.53,p = .170). However, on day 2, the LEC lesion groupwas performing above chance (t(5) = 2.91,p = .034) while the shams remained at chancelevels of accuracy (t(7) = 1.43,p = .197). Shams reached above-chanceperformance by day 3 (t(7) = 7.51,p < .001), matching the lesion group(t(5) = 2.74,p = .041). Both groups showed above-chance accuracyon training days 3 to 7.With the inclusion of the unilateral LEC lesion animal, a 2 × 7 mixedANOVA revealed a significant main effect of day = 3.23,p = .039, F = 0.16,p = .701, F = 1.24,p = .311, M = –8.77, SE = 1.98,p = .021). These results indicate that both theLEC lesion animals and shams were similarly motivated and decisive incompleting the spatial task. In addition, the change in latencybetween the first and last training day for both groups suggests thatanimals were learning the task over time, as expected. Thesignificance of the data did not change when the animal with theunilateral lesion was included.Response latencies were compared between the two groups across trainingdays as a proxy for evaluating decision-making certainty andmotivation towards the food reward. eraction . Post hoThe hippocampal–entorhinal network has been shown to be critical for spatialmemory . RecentOn first inspection, this argues against the suggestion that LEC ispreferentially involved in processing egocentric rather than allocentricspatial information and rather suggests that the principle role of LEC isthe integration of features of an event. However, closer inspection of thedata reveals that while LEC lesioned rats are impaired at the allocentricversion of the OP task, relative to shams, they are still performing abovechance. Interestingly, LEC lesioned animals’ ability to recognise a familiarOP association within an allocentric spatial framework took longer todevelop than controls with significant memory for OP associations onlyoccurring in the second half of the allocentric OP test trials. This showsthat they can still use an allocentric frame of reference to remember theassociation of object with location even if it is not as efficient a processas it is in controls. In contrast, performance of the LEC lesioned rats onthe egocentric version of the task is at chance. Clearly, while LEC iscritical for the association of object and place, it is particularlyimportant for tasks that involve processing of spatial information in anegocentric frame of reference.How is it possible for rats with LEC lesions to remember the allocentriclocation of a previously experienced object? Previous experiments usingcomplete LEC lesions had shown deficits in all associative recognitionmemory tasks , 2013b. We went onto examine whether lesions of LEC would produce a general deficit inspatial memory by examining performance on a non-associative referencememory task on the T-maze. The data show that rats with lesions of the LECwere not impaired and were actually better than controls in the early stagesof the task. It is possible for rats to use either egocentric or allocentricframes of reference to solve the T-maze task and so it is interesting toexamine whether the associative recognition tasks give an insight into howLEC lesions affected rats’ ability to remember the correct spatial locationon the T-maze. Results from the OP experiments show that LEC animals do notremember the egocentric position of a previously seen object but do rememberits allocentric position, albeit less well than shams. One interestingpossibility here is that normal animals spend the first few days of T-mazetraining learning both an egocentric and allocentric frame of referencewhile LEC lesioned rats rely solely on the allocentric. This would mean theyhave less information to learn, making the task easier which could explainthe increased T-maze accuracy in the LEC lesioned rats. Future studies couldincorporate rotation trials where allocentric and egocentric frames ofreference are placed into conflict to examine whether disruption of LECchanges strategy use.Previous studies have suggested that LEC may process local spatial informationrather than distal, global cues. We have previously shown that LEC lesionedrats can remember the position of food rewards in relation to globalallocentric space but are impaired when learning the position of foodrelative to local cues . The curThe current data are consistent with unit recording data that have shown LECresponses to objects within the environment . ThesesPrevious studies have shown that LEC is one of four cortical hubs whichreceives extensive connections from the rest of cortex putting Overall, the current findings are in agreement with previous studies suggestingthat the central role of LEC is in the integration of features that make upepisodic memory , 2013b.


Adding optical protection from water and high humidity reduces these values by ~10%. We describe tradeoffs between peak efficacy and cost.Light-emitting diodes (LEDs) have enabled a historic increase in the conversion of electric energy to photons, but this is approaching a physical limit. The theoretical maximum efficiency occurs when all input energy is converted to energy in photosynthetic photons. Blue LEDs can be 93% efficient, phosphor-converted “whites” 76% efficient, and red LEDs 81% efficient. These improvements open new opportunities for horticultural lighting. Here we review (1) fundamental physics and efficiency of LEDs, (2) the current efficacy of LEDs, (3) the effect of spectral quality on crop yield, and (4) the potential efficacy of horticultural fixtures. Advances in the conversion of photons to yield can be achieved by optimization of spectral effects on plant morphology, which vary among species. Conversely, spectral effects on photosynthesis are remarkably similar across species, but the conventional definition of photosynthetic photons (400–700 nm) may need to be modified. The upper limit of LED fixture efficacy is determined by the LED package efficacy multiplied by four factors inherent to all fixtures: current droop, thermal droop, driver (power supply) inefficiencies, and optical losses. With current LED technology, the calculations indicate efficacy limits of 3.4 µmol JThe term efficiency applies to ratios with the same units in the numerator and denominator, which can be expressed as a percentage. LED efficiency describes the optical power output divided by the electrical power input (watt/watt or %). The term efficacy applies to ratios with different units. In horticultural lighting, efficacy refers to micromoles of photon output per second, per watt of input power. Since a watt is a joule per second, this simplifies to µmol per joule. The relationship between photon energy and wavelength is expressed in the Planck–Einstein relation, often just called Planck’s equation. This equation states that energy is inversely proportional to wavelength . LED performance specifications are for LED packages. An LED fixture refers to LED packages integrated into a fixture.]By converting LED efficiency into efficacy, we get the appropriate units for determining the impact of photons on plants per input electrical power. This follows another physical law called the Stark–Einstein Law, which states that for every photon absorbed, only one molecule can react. This Law can be restated to say that one photon excites one electron. In this paper, photon efficacy is limited to photons between 400 and 700 nm, except in the case of far-red LEDs, where photons from up to 800 nm are included. LED package manufacturers often report efficacy in lumens per watt, because this is a meaningful metric for human lighting, but it is not applicable for horticultural lighting because it is a measure of photons weighted for human vision based on the human eye response to different colors.photon/Vf), affected by internal electrical resistance of the LED.Electrical efficiency: the ratio of the emitted photon energy expressed in electron volts to the applied voltage : the conversion of electrons to photons, affected by non-radiative recombination pathways, including impurities and microphysical defects.Photon extraction efficiency: the ratio of photons that exit the LED semiconductor material to total generated photons, affected by internal reflection and reabsorption. Losses in extracting photons out of an LED package are termed “package losses” within the LED industry. These can vary greatly among LED package types.The fundamental efficiency of LEDs (LED packages) is the product of the following three sub-efficiencies:1. Incremental improvements have been made to each of the three factors above resulting in a substantial improvement of LED packages over the past 10 years. Now, far-red, red, white, and blue LEDs, respectively, can be 77, 81, 76, and 93% efficient  that absorbs blue photons and luminesces at longer wavelengths. Phosphor-converted white LEDs are designed to transmit some blue photons, with the remainder converted to longer wavelengths. Types and amounts of phosphor are varied to create multiple hues and color qualities. Figure 2). Both CRI and the TM-30 metric of Rf use a scale of 0–100 to describe color fidelity. A high color fidelity facilitates observing subtle color differences. This is important to human observers for visual identification of tiny insects, nutritional disorders, and diseases. Suitable color fidelity is also necessary for machine vision.White LEDs consist of blue LEDs with a luminescent material coating , neutral white (3500–4500 K), cool white (4500–5500 K), and daylight (5500–7500 K). A lower CCT (2700–4000 K) and higher CRI (greater than 80) are often preferred for indoor lighting to provide incandescent-like light qualities for humansIncreased density of phosphor coatings and increased use of red phosphor materials decreases efficiency. A 6500 K LED (daylight) with about 30% blue photons can have 95% of the photon output of its non-phosphor-converted blue LED counterpart, but this value decreases to 80–85% for a warm white LED with 10% blue photons. In addition, as the optical output of white LEDs increases, the phosphor efficiency will decrease. This is due to conversion, energy, and optical losses within the phosphor conversion process.LEDs are designed for performance at specific current ranges. Moderate LED drive current density enables higher efficiency, but at very low drive currents, efficiency decreases. LED loss mechanisms are typically a function of current density. So at a given current, increasing the size of an LED chip can increase the efficacy of an LED by reducing the current density. Unfortunately, chip area is often confidential, and LED manufacturers only report LED specifications at the total LED drive current, not drive current density.4, Osram 7, and Samsung 8. These companies provide LED efficacy data in µmol J−1. Additional LED manufacturers include Nichia 9, Cree 10, Epistar 11, and many others. As technology improves, see the companies’ websites to find the latest LED package efficacy information, and apply the principles described below to determine potential fixture efficacy. Figure 13. For a white LED, which has a blue LED and a phosphor conversion layer, the droop is caused by Auger in the blue LED and reduction in phosphor conversion efficiency at higher optical flux concentrations14. For red and far-red LEDs, the current droop is caused by carrier leakage due to poor confinement of electrons in the active regions15. In general, decreasing the drive current increases the efficacy, but eventually Shockley–Read–Hall defect losses will dominate at very low drive current16. LED manufacturers continue to both increase LED peak efficiency and reduce current droop. The theoretical maximum lines for red (centered at 660 nm) and blue (centered at 450 nm) LEDs are based on the assumption of 100% power efficiency of the LED or hypothetical photon generating device (1 W electricity input = 1 W photon output) followed by a conversion to number of photons using Planck’s equation. White LEDs would be as efficient as blue LEDs if phosphor conversion was 100% efficient. However, phosphor conversion efficiencies range from 80 to 95%, depending on amount of phosphor, phosphor material, temperature, and photon flux density. A color-mixed “white” fixture using direct-emitting (not phosphor converted) green LEDs (550 nm) as well as red and blue LEDs would have a theoretical maximum of ~4.6 µmol J−1, but direct-emitting green LEDs currently have low efficiency (referred to as the green gap17) resulting in low efficacy (about 1.9 µmol J−1)18.Figures Junction temperature refers to the operating temperature at the actual diode. There are two temperature standards for reporting the efficacy of LEDs: 25 and 85 °C. Efficacy decreases ~10%, as the temperature increases from 25 to 85 °C . Thermal droop is typically worse in red compared with blue LEDs.19).A timeline of the historic and projected increases in LED efficacy is presented in Fig. Figure Horticultural LED fixtures typically contain combinations of red (peak ≈ 660 nm), blue (peak ≈ 450 nm), white, and/or far-red (peak ≈ 730 nm) emitting LEDs. Other peak wavelengths are available, but they have lower efficiencies and efficacies and are less common. Fixture manufacturers choose the ratios of these LEDs for specific applications and based on their perception of best practices and market demands.20. The first LED-produced photons used to grow plants were red21, which was shortly followed by the development of high output blue LEDs22. For a review of the historical significance of this Nobel prize-winning discovery, see ref. 23. Before widespread adoption of blue LEDs, early studies demonstrated the value of blue photons for plant growth using blue fluorescent fixtures to supplement red photons from LEDs25.Morrow discussed the significance of LEDs for horticultural lighting and reviewed the early adoption of the technology27. Due to widespread use in lighting applications for human vision, white LED packages are now ~20% of the cost of red LEDs. This has contributed to the increase in the fraction of white LEDs to more than 60% in some horticultural fixtures.The first commercial horticultural LED fixtures were blue + red combinations. These fixtures, which produced a spectrum that appeared magenta, had a higher efficacy than white or white + red fixtures. Many people thought that these blue + red fixtures would enhance photosynthesis compared with full spectrum fixtures due to their close match to the chlorophyll absorption spectrum, which shows peak absorption in the blue and red regions of the photosynthetically active radiation  spectrum. This thinking was advanced by early LED manufacturers, even though green photons have long been known to be effective for photosynthesisPhotons excite electrons and photobiology is thus driven by the number of photons, not energy or lumens. Biologically active photons must have sufficiently high energy to excite pigment photoreceptors, and there are multiple photoreceptors with weighting functions for wavelengths, which are biophysically or empirically derived. Lumens are an example of a weighting function applied to a photon flux and spectral distribution for human visual function.28, and may vary over the plant life cycle. Some principles, however, apply across all species. The impacts of spectrum on plant growth and development are much greater in sole-source lighting than in greenhouse supplemental lighting where electric lighting makes up only a small portion of the plant lighting diet.The effect of spectral quality on plant shape is synergistic among wavelengths, interacts with intensity, varies among speciesIn plant biology, spectra are traditionally separated into the following coarse categories.Ultra-violet photons are further separated into three broad categories: UV-C (100–280 nm), UV-B (280–315/320 nm), and UV-A (315/320–400 nm). The wavelength at which UV-C and UV-B are separated (280 nm) is determined by the shortest wavelength of solar radiation that reaches the surface of Earth. The wavelength at which UV-B and UV-A are separated (315 or 320 nm) is generally determined by the effect of sun on human skin sunburn (315 nm) or skin cancer (320 nm). There is no universal agreement on the wavelength transition between UV-B and UV-A, both are equally used. Fortunately, UV-C photons are completely blocked by our atmosphere because they are highly damaging to biological organisms. UV-B photons are also damaging, but can have beneficial effects including increased production of secondary metabolites29. UV-A photons are less damaging than UV-B, and can have either stimulatory or inhibitory effects on plant growth, depending on species and interacting environmental factors30.31, but these photons can have large biological effects in small quantities. At 25 °C and 700 mA, the efficiency of UV-A LEDs increases from 50 to 60% as the wavelength increases from 370 to 395 nm32. A violet LED with a peak between 402 and 408 nm is ~65% efficient, and has 15–30% of its photons below 400 nm. Efficiency will increase as current density decreases.At 25 °C and 350 mA, UV-B and UV-C LEDs are only ~3% efficient27, PAR only includes photons with wavelengths between 400 and 700 nm. However, McCree’s studies show significant differences in the photosynthetic efficiency of species at wavelengths below ~425 nm. Some species, like radish, have equal photosynthesis between 375 and 500 nm. Photons between 350 to 400 nm can be photosynthetic, but a high fraction are typically absorbed by non-photosynthetic pigments.Based on studies by McCreeBlue photons (400–500 nm) reduce plant height and leaf expansion in nearly all species35. Because of absorption by inactive pigments , blue photons are ~20% less photosynthetically efficient than photons from the most common red LED (660 nm)27. However, the blue-induced decreases in leaf area (reducing photon capture) may have a larger effect on overall plant growth than the blue-induced reduction in photosynthetic rate28. A range of 5–30% blue is typically used in horticultural LED fixtures to inhibit excessive stem extension and reduce plant height, which is typically beneficial for controlled environment growth.Green photons (500–600 nm) improve human perception of color. Unfortunately, monochromatic direct emitting (non-phosphor converted) green LEDs have low efficacy. White (phosphor-converted blue) LEDs are thus used to provide the green photons that are important to human vision; and they have the added benefit of providing blue and red photons. Green photons are up to 10% less photosynthetically efficient than photons from the most common red LED (660 nm)27, but they penetrate deeper into plant canopies than blue or red photons36.Arabidopsis suggest that green photons can reverse blue photon effects 38 or induce shade avoidance 40. Some studies suggest that green-induced shade avoidance also occurs in food crops and other economically valuable plants41, but several other studies have shown minimal effects45.The effect of green photons on plant shape is generally much less than the effects of blue or far-red photons. Studies in Red photons (600–700 nm) are well absorbed by leaves, are photosynthetically efficient, and are efficiently generated by LEDs so they are widely used in horticultural fixtures. The classical paradigm has been that red and far-red act antagonistically to inhibit or induce shade avoidance symptoms, such as stem elongation, hyponastic leaf orientation, and/or reduced branching47. However, the high absorbance of red photons by chlorophyll means that the impact of red on shade responses may be overestimated48. Replacing green photons with red photons has minimal effects on plant shape45, but plants grown in the complete absence of red and green photons (sole-source blue LEDs) can rapidly elongate49.Far-red photons (700–800 nm) can have powerful effects on plant shape, and are efficiently generated by LEDs so they are a promising addition to horticultural lighting. Along with several other laboratories, we are working to quantify the effects of far-red photons on plant morphology. In some species , far-red photons beneficially increase leaf expansion, but they also significantly increase stem elongation in many other species50, which may not be beneficial.52. These photons are thus being reconsidered for their role in photosynthesis. Far-red photons must be used with caution, particularly in sole-source environments, because they can induce stem elongation associated with shade avoidance.Despite the classic definition of PAR, recent studies indicate that far-red photons (700–750 nm) are photosynthetically synergistic with shorter wavelength photons53:LED drive currentLED junction temperatureDriver efficiencyOptical losses in the fixtureThe upper limit of fixture efficacy is determined by the choice of LEDs and operating conditions. Different colors of LEDs have different efficacies, but the quality of LEDs within and among manufacturers also varies. In addition to the efficacy of the selected LEDs, fixture efficacy is determined by four additional factors1. Drive currentThe effect of drive current on LED efficacy has been discussed above. Due to different types of efficiency reductions at high and low drive currents, efficiency can be maximized at some relatively low drive current (less than 100 mA). Fixture manufacturers seek to co-optimize fixture size, cost, output, and efficacy for specific applications. While low drive currents will increase the efficacy of the fixture, the output of each LED would be relatively low. This increases the cost and complexity of the fixture.2. LED junction temperature53. Better thermal management may increase fixture cost, but it also increases efficacy and longevity of the LEDs . A typical L70 for LED fixtures is 50,000 h. As the LEDs and fixture age, the efficacy will decline; a problem that is exacerbated by high junction temperatures. Rates of fixture aging can vary greatly among manufacturers. Most manufacturers characterize their fixture lifetime  in terms of LED output depreciation based on a standard LED package test—IES LM-80, which can be interpolated into luminaire lumen maintenance. Projections of luminaire lumen maintenance based on LED depreciation cannot exceed six times of the duration that the LEDs were tested, so for a depreciation lifetime claim of 60,000 h the LEDs must have been tested for 10,000 h. Many fixtures that claim extended lifetimes are exceeding the allowable six times interpolation based on LED testing. Fixture lifetimes based on LED depreciation also do not include optical loss mechanisms in the fixture and accelerated aging of the LEDs due to higher temperatures. Also, typical lifetime claims do not consider catastrophic failure of the LED driver, which often fails before the LEDs have reached the L70.]The junction temperature of LEDs in fixtures depends on the drive current, ambient temperature, and the heat dissipation  of the fixture, but is typically around 85 °C3. LED drivers  are necessary to convert AC to DC power and provide regulated voltage and current. The efficiency of LED drivers ranges from 85 to 95%. LED drivers can be less efficient when they provide dimming, color control and/or communication functionality.4. Optical losses occur when LEDs are mounted in fixtures. The sides of the fixture can obstruct low-angle photons. Protective transparent covers  transmit up to 92% of the photons and thus reduce the output by 8%, but this protection can significantly improve the lifetime of a fixture. Fixtures with unprotected LEDs can have 99% optical efficiency, but may have shorter lifetimes in harsh growing environments .56.Optical covers can also diffuse the photons, which reduces efficiency, but can result in more uniform mixing of colors and improved photon penetration into plant canopies57.Photons must impact leaves to be absorbed, and this is an important consideration in fixture design. Early LED fixtures had focused photon output over a small area. This facilitated precise photon placement, but caused nonuniform distribution. LED package and fixture design has transitioned to a less-focused photon distribution, but as long as the photons exit the fixture this does not affect our analysis of optical efficacy−2 to 50 mA mm−2).Drive current minimized to achieve 104% of the reported LED efficacy  to achieve 95% of the reported LED efficacy (at 25 °C).The LED driver is 95% efficient.Unprotected LEDs in the fixture to achieve 99% optical efficiency.Using the following near-maximum parameters, we now calculate an achievable fixture efficacy using presently-available technology:−2 and 25 °C here). Reducing drive current is much easier than reducing temperature.The resulting fixture efficacy would be 1.04 × 0.95 × 0.95 × 0.99 = 93% of the reported efficacy of the LED. The first two factors can be above 100%, if the LEDs are operated at lower drive current and lower temperature than the reported specification  could potentially achieve an efficacy of 4.1 µmol J−1 .The LED driver is 90% efficient.Protected LEDs in the fixture achieve 92% optical efficiency.Using more typical parameters:The resulting fixture efficacy would be 0.90 × 0.90 × 0.90 × 0.90 = 67% of the reported efficacy of the LEDs.−1 (ref. 57).In 2014, the best LED fixtures had an efficacy of 1.7 µmol J−1 for white + red fixtures and 3 µmol J−1 for blue + red fixtures have been achieved  maintains a list of horticultural lighting products that meet their listing requirements−1 of output capacity. HPS fixtures (1 kW) range from $200 to $350 USD per kW (5–9 µmol s−1 per $). LED fixtures range from $1000 to $3000 USD per kW (0.5–1.5 µmol s−1 per $). On a photon flux basis, the initial cost is therefore, 3–18 times higher for LED vs. HPS fixtures. The cost of both technologies has decreased, but the cost of LED fixtures are expected to decrease faster than HPS fixtures. While initial cost is higher, LED fixtures reduce energy cost compared with HPS. Depending on usage periods and price of energy, the electric savings can equal the difference in initial cost after of 3–5 years for sole- source applications and 5–8 years for supplemental applications57.Initial fixture cost per photon s59.Spectral quality for plant morphology and photon capture60.Adequate green photons to create “white” light to facilitate human comfort and visual identification of insects, diseases, and nutritional disordersFixture reliability, including environmental protection of the LEDs.Fixture operating temperatures, which affect LED and system longevity.Uniformity and distribution of photon output. Many early LED fixtures had narrow beam angles, but more recent fixtures have a broader distribution of photons. High wattage HPS fixtures need to be mounted higher above the canopy than LED fixtures to achieve uniform distribution of photons.Fixture size for shading in a greenhouse application.In addition to efficacy, several other factors affect fixture choice, including:62. However, some recent evidence indicates that longer-term cycling of LEDs (minute to hours) can alter plant shape (hypocotyl length) and color (anthocyanin synthesis)63.Because LEDs can be cycled on and off over short intervals, there has been interest in rapid cycling of LED fixtures to improve plant growth. Unfortunately, high frequency flickering (10–10000 Hz) has been well-studied, and there is neither empirical nor theoretical evidence that this can be used to increase the quantum yield of photosynthesis. Plants appear to integrate light intensity for photosynthesis−2 and a junction temperature of 25 °C.Blue LEDs are now 93% efficient, phosphor-converted “whites” are 76% efficient, and reds are 81% efficient when run at the near-optimal conditions of 100 mA mmBoth junction temperature and drive current density will affect the photon efficacy of LEDs, and in general, the most efficient LED fixture will run their LEDs at low drive currents. However, a lower drive current results in a lower photon output per LED, and the resulting fixture will require many LEDs to achieve a high photon output and thus will be more expensive.Broad spectrum distribution of photons is useful for diagnosis of plant disorders. Broad spectrum lighting is not necessarily beneficial for photosynthesis or plant growth. Unique spectra, selectively applied during specific stages of the life cycle, can, however, have a beneficial effect on plant shape and development.−1 for white + red fixtures, and 4.1 µmol J−1 for blue + red fixtures. These values are significantly higher than current typical values of 2–3 µmol J−1.The calculations in this paper show current possible performance levels of LED fixtures of 3.4 µmol Jhttps://cubes.space/; https://www.hortlamp.org/index.html.Although fixture efficacy is paramount, timing and angular delivery of photons to photosynthetic tissues, spectrum, and intensity also determine the effectiveness of the photon delivery system. Efficient lighting is then coupled with optimal temperature, humidity, nutrition, plant water potential, atmospheric carbon dioxide concentration, delivery of oxygen to root surfaces, and genetics. Both NASA and the USDA are funding research at universities to optimize these factors and improve the economic potential of electric lighting in in controlled environments:


Recent reports suggest SAA is sufficient to induce atrophy via TLR. Therefore, we assessed TLR2,4, and IL‐6 mRNAs in hindlimb muscles. TLR mRNAs were not altered, suggesting SAA effects on atrophy during LLC are independent of TLR signaling. However, we noted > 6‐fold induction of IL‐6 in soleus of HU mice, despite minimal shift in the plasma proteome, indicating potential localized inflammation in atrophying muscle. Furthermore, paraoxonase 1 (PON1) was highly repressed in LLC mice and largely undetectable by immunoblot in this group. Our data suggest SAA and PON1 as potential novel atrokines for cancer cachexia and indicate localized inflammation in atrophying muscles independent of the plasma proteome.Skeletal muscle atrophy is common across a variety of pathologies. Underlying mechanisms of atrophy differ between pathologies, and in many conditions, circulating factors are tied to muscle atrophy. Therefore, we sought to identify alterations to the plasma proteome across divergent forms of muscle atrophy, disuse and cancer cachexia, as potential mediators of atrophy. C57BL6/J mice were assigned to Lewis Lung Carcinoma (LLC)‐induced cachexia, disuse by hindlimb unloading (HU), or control (CON). Plasma samples were submitted for discovery proteomics and targets of interest confirmed by immunoblot. Considerably more peptides were altered in plasma from LLC (91) than HU (9) as compared to CON. Five total proteins were similarly modulated in HU and LLC compared to CON, none reached criteria for differential expression. Serum Amyloid A1 (SAA) was 4 and 6 Log Skeletal muscle atrophy is a debilitating condition associated with increased morbidity and mortality. In this study, bloodborne factors were assessed via plasma proteomics to identify candidates which may contribute to the development of this condition in disuse and cancer cachexia. We identify Serum Amyloid A as a factor heavily induced in cancer cachexia which may contribute to muscle atrophy in the tumor‐bearing state. Performing such global plasma proteomic analysis could lead to the identification of novel secretory pro‐atrophic factors (atrokines) and potentially reveal overarching targets linking the two forms of atrophy. Therefore, the purpose of this study was to compare and contrast the plasma proteomes between pre‐clinical models of cancer‐cachexia and disuse‐induced atrophy to identify novel secretory atrokines as potential markers and mechanisms of muscle atrophy.22.1All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Arkansas, Fayetteville. In total, 34 male and 42 female C57BL/6J mice were purchased from Jackson Laboratories . The animals were housed in a temperature‐controlled room and maintained on a 12:12 hr light‐dark cycle. The mice were given ad libitum access to normal rodent chow and water.In this study, we utilized two different pre‐clinical models of muscle atrophy—hindlimb unloading (HU) to recapitulate disuse‐induced atrophy and Lewis Lung Carcinoma (LLC) implantation to induce cancer cachexia. A cohort of control (CON) animals was age matched to both experimental groups. All animals were singly housed to match housing requirements of HU. This design created three experimental groups: CON, LLC, and HU which were then duplicated to represent both male and female mice. Experiments were performed such that animals in all experimental groups were euthanized and tissue collected at 11–12 weeks of age in order to age‐match groups at time of tissue collection. At the end of designated interventions, animals were anesthetized under isoflurane, body weight recorded and animals humanely euthanized. Plantaris, gastrocnemius, soleus, extensor digitorum longus (EDL), tibialis anterior, quadriceps, spleen, epididymal fat, testes, and plasma were quickly collected post‐euthanasia. Tissue samples were weighed and snap‐frozen in liquid nitrogen for further processing and stored at −80ºC. Plasma samples were centrifuged at 3,500 g for 15 min. Plasma was pipetted into separate sample tubes and stored at −80ºC. One hundred microliterof plasma was aliquoted and sent to the University of Arkansas Medical Sciences TRI Proteomics Core for discovery proteomics (detailed below).2.26) suspended in 100 µl sterile PBS and injected subcutaneously into the hind flank of mice at 8 weeks of age as previously described  was dispensed on a microscope slide. Slides were air‐dried overnight to allow for cell adhesion to the microscope slide. Slides were stained with 0.1% crystal violet stain  as per the manufacturer's instructions. Tandem mass tag (TMT) proteomics was performed off‐site  software was used for canonical pathway analysis, upstream analysis, and network discovery.Ingenuity Pathway Analysis  were thus identified as surpassing threshold levels of 33.1p > .05). Total HU body weight was significantly lower (8%) than CON (p < .05). However, tibia length, an indicator of total body size independent of weight, was not different between experimental groups. Therefore, as total body size was not different tissue weights are presented as raw, non‐normalized averages. In both LLC and HU groups, plantaris and gastrocnemius, weights were significantly lower compared to CON (9%–23% p < .05). However, these weights were not significantly different between atrophic conditions (p > .05). Soleus weight was lower in HU compared to CON and LLC , while LLC soleus weight was only significantly lower compared to CON . Spleen wet weight, a surrogate marker of inflammation, was ~ 200% greater in the LLC animals compared to CON and HU (p < .05) (Table Phenotypic descriptors of applicable body and tissue weights are presented in Table p < .05). However, female LLC mice did not display significant differences in muscle masses compared to CON despite significant tumor mass, and compared to CON lower gonadal fat mass  and elevated spleen mass . Prior data have demonstrated that female mice typically become cachectic only after becoming acyclic  and tested estrous cycle throughout, however, none of the mice in the current study became acyclic in agreement with the lack in measurable muscle wasting in these mice  or in extracted table (10.6084/m9.figshare.12994511). Using TMT‐based discovery proteomics, a total of 368 Protein IDs were identified in the plasma of the male animals in this experiment. Among these, 5 were similarly modulated between LLC and HU groups and were significantly different compared to CON, though none reached DE thresholds  from CON. Total number of DE peptides discovered in each comparison of groups are presented in Table All proteomics data are deposited in Figshare and can be accessed as Scaffold Viewer file (10.6084/m9.figshare.12994496).To determine function and relations between identified peptides we next performed pathway analysis by IPA to determine signaling pathways most affected by shifts in the plasma proteome during muscle atrophy, this analysis was designed to provide insight to the function of identified proteins prior to more detailed examinations. Here we present the top 5 modulated pathways in each comparison Figure , detaile3.32FC: 4.17 LLC, −2.16 HU compared to CON). Apolipoproteins, Fibrinogens, SerpinA3, SerpinF1, PON1, antioxidant proteins, and others were found to be predominantly modulated in LLC plasma. Similarly, SAA, SerpinA1, and Fibrinogens were predominantly modulated in HU plasma.In Figure 3.4p > .05). However, mean SOD3 content was ~ 60% greater in HU plasma compared to LLC . SOD3 was chosen as a surrogate of antioxidant proteins that has previously been observed to be readily induced by physical exercise and to protect against some forms of muscle atrophy  in spleen, kidney and lung. In liver, despite high expression (Ct ~ 21) no induction was observed in LLC mice. SAA1 expression was observed in LLC tumors with a Ct mean of 35. Due to the lack of significant effects these data are not shown.SAA1, TLR2, TLR4, MuRF‐1, Atrogin‐1, IL‐6, and TNF‐α in the muscle as previously observed  but not in the un‐atrophied EDL  which also displayed the greatest degree of muscle atrophy  and HU (>6 fold) compared to CON as well as of TNF‐α in the soleus only (2.7‐fold). To further assess TLR/NFΚB signaling we assessed mRNA contents of NFΚB1 and NFΚB2. Both isoforms of NFΚB were elevated in soleus of HU mice, by 2.3 and 1.7‐fold, respectively, similar to IL‐6 and TNF‐α, with no significant effects observed in LLC mice.A recent work by Hahn et al.  across muscle wasting. We demonstrate large shifts in the plasma proteome in mice undergoing cancer cachexia with few shifts in plasma peptides under conditions of disuse. Specifically, we note the large induction of SAA1/2 in the plasma of tumor bearing mice. Notably, SAAs are sufficient to induce muscle atrophy . In contrast, only nine peptides reached criteria to be considered DE when comparing HU to CON. This demonstrates the differential reliance of the two forms of muscle atrophy on the plasma proteome. Interestingly, though no peptides were similarly altered between LLC and HU conditions compared to CON which reached the DE threshold, five peptides were similarly altered between conditions and met the statistical significance requirement. While purely speculative at this point these five peptides Figure  may prov4.3SAA1 was first described as an inducible factor in plasma during cancers in 1993  where are they produced in LLC tumor‐bearing mice?, and (b) is there evidence SAAs are working through TLR‐induced signaling to promote muscle atrophy in tumor‐bearing mice? To answer the first question we assessed SAA mRNA across tumor, lungs, liver, spleen, kidney, and muscle. Across these tissues we observed no induction of SAA in the LLC mice. However, SAA was expressed by the tumor (Mean Ct ~ 35), a source absent in CON and HU mice. We should do note a prior micro‐array analysis by Bonetto et al.  is sufficient to protect from atrophy induced by dexamethasone or calsequestrin expression induced heart failure  which may suggest a role for localized inflammation during disuse inflammation.The authors declare no conflict of interest.NPG and TAW conceived and designed the research. SL, KRD, MERC, WSH, LTJ, TAW, and NPG performed the experiments. SL and KRD analyzed the data. SL, KRD, and NPG interpreted the results of the experiments. SL, KRD, and NPG prepared figures. SL, KRD, and NPG drafted the manuscript. SL, KRD, MERC, WSH, LTJ, TAW, and NPG edited and revised the manuscript. SL, KRD, MERC, WSH, LTJ, TAW, and NPG approved the final version of the manuscript.


Nintedanib is an approved therapy for idiopathic pulmonary fibrosis (IPF). Some patients treated with nintedanib experience weight loss. Exploratory data suggest that low body mass index or weight loss are associated with worse outcomes in patients with IPF. We investigated whether BMI at baseline or weight loss over 52 weeks was associated with FVC decline, or influenced the effect of nintedanib, in patients with IPF.2) and by weight loss over 52 weeks  using random coefficient regression.Using pooled data from the two INPULSIS trials, we analysed the rate of decline in FVC (mL/yr) over 52 weeks in patients treated with nintedanib and placebo in subgroups by baseline BMI . Nintedanib reduced the rate of FVC decline versus placebo in all subgroups by BMI, with a consistent treatment effect across subgroups (interaction p = 0.31). In the placebo group, the mean rate of FVC decline was numerically greater in patients with > 5% than ≤5% weight loss over 52 weeks (− 312.7 [SE 32.2] versus − 199.5 [SE 14.4] mL/year). Nintedanib reduced the rate of FVC decline versus placebo in both subgroups by weight loss, with a greater treatment effect in patients with > 5% weight loss (interaction p = 0.0008). The adverse event profile of nintedanib was similar across subgroups.In the placebo group, the mean rate of FVC decline over 52 weeks was numerically greater in patients with lower baseline BMI (− 283.3 [SE 22.4], − 207.9 [20.9] and − 104.5 [21.4] in patients with BMI < 25 kg/mIn patients with IPF, lower BMI and weight loss may be associated with faster decline in FVC. Nintedanib reduces the rate of FVC decline both in patients who lose weight on treatment and those who do not.ClinicalTrials.gov; Nos. NCT01335464 and NCT01335477; URL: www.clinicaltrials.gov. Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterised by progressive loss of lung function . IPF maiSome studies have suggested that lower body mass index (BMI) –7 or weiNintedanib is a tyrosine kinase inhibitor approved for the treatment of IPF. In the two Phase III 52-week INPULSIS trials, nintedanib reduced the rate of decline in FVC in patients with mild or moderate impairment in lung function by approximately 50%, with an adverse event profile characterised mainly by gastrointestinal events . Weight The INPULSIS trials (NCT01335464 and NCT01335477) were two randomised, double-blind, placebo-controlled trials of nintedanib in patients with IPF, performed at 205 sites in 24 countries. Eligibility criteria for the INPULSIS trials have been described . BrieflyPatients were randomised 3:2 to receive nintedanib 150 mg twice daily or placebo for 52 weeks, with a follow-up visit 4 weeks after treatment discontinuation. Treatment interruption and dose reduction to 100 mg twice daily were allowed to manage adverse events. After an adverse event had resolved, the dose could be increased back to 150 mg twice daily. Patients who discontinued study drug prematurely were asked to attend all scheduled visits and undergo all examinations as originally planned. FVC was measured at baseline and at weeks 2, 4, 6, 12, 24, 36, and 52 using sponsor-provided spirometers. Weight was measured at baseline and at weeks 2, 4, 6, 12, 24, 36, and 52.The pre-specified subgroup analyses of the INPULSIS trials have been reported , 16. Her2) or weight loss over 52 weeks  to the model. In addition, we assessed outcomes in subgroups by BMI at baseline  and by weight loss over 52 weeks . Weight loss ≤5% or > 5% over 52 weeks was assessed based on the annual rate of decline in weight analysed using a random coefficient regression model with fixed effects for trial, treatment, sex, age and height and random effect of patient specific-intercept and time. We also analysed the annual rate of decline in FVC (mL/year) in subgroups based on BMI below and at least the median at baseline, and in subgroups by weight loss over 52 weeks  based on the change from baseline in weight at week 52 . In analyses of the annual rate of decline in FVC, the term subgroup and the interaction terms treatment-by-subgroup, time-by-subgroup and treatment-by-time-by-subgroup were included in the model. The model allowed for missing data, assuming that they were missing at random; missing data were not imputed. The interaction p-values are an indicator of the difference in the effect of nintedanib versus placebo among the subgroups. The other statistical analyses are described in Supplemental Appendix Analyses were conducted using data from patients who received ≥1 dose of nintedanib or placebo. In the primary analysis, the annual rate of decline in FVC (mL/year) was analysed using a random coefficient regression model (with random slopes and intercepts) with fixed effects for trial, treatment, sex, age and height and random effect of patient-specific intercept and time. To assess the effect of BMI at baseline or weight loss on the treatment effect of nintedanib, we repeated the original analyses with the addition of covariates for race  and either BMI at baseline .A total of 638 patients with IPF were treated with nintedanib and 423 with placebo. Baseline characteristics were balanced between the nintedanib and placebo groups. Most (79.3%) of the participants were male. Mean (SD) age at baseline was 66.8 (8.0) years, weight was 79.0 (16.6) kg, BMI was 27.9 (4.6) kg/m2, ≥25 to < 30 kg/m2 and ≥ 30 kg/m2, respectively. Compared with patients with BMI ≥30 kg/m2, greater proportions of patients with BMI < 25 or ≥ 25 to < 30 kg/m2 were of Asian race, and greater proportions with BMI < 25 kg/m2 had never smoked , 453 (42.7%) and 301 (28.4%) patients had BMI < 25 kg/m2 (− 283.3 [SE 22.4]) than BMI ≥25 to < 30 kg/m2 (− 207.9 [20.9]) or BMI ≥30 kg/m2 (− 175.7 [27.0])   . Similar results were observed when the rate of decline in FVC was analysed in subgroups by baseline BMI above and below the median (27 kg/m2)  and race were included in the model analysing the rate of decline in FVC (mL/year) over 52 weeks, the difference between treatment groups was consistent with the original analysis . The proportions of placebo-treated patients who had an investigator-reported acute exacerbation was similar across these subgroups , while the proportions of patients who died was numerically greater in patients with baseline BMI < 25 kg/m2 than ≥25 to < 30 kg/m2 or ≥ 30 kg/m2 .The mean increase (worsening) in SGRQ total score at week 52 in placebo-treated patients was numerically greater in patients with baseline BMI < 25 kg/m2 than ≥25 to < 30 kg/m2 or ≥ 30 kg/m2, but the p-values for treatment-by-subgroup interaction did not indicate heterogenous treatment effects across the subgroups (Table Differences between the nintedanib and placebo groups in absolute changes from baseline in FVC (mL and % predicted) and change in SGRQ total score were numerically greater in patients with BMI < 25 kg/mps Table . Differeps Table .The mean (SE) annual rate of decline in weight over 52 weeks was − 3.3 (0.2) kg/year and − 1.5 (0.2) kg/year in the nintedanib and placebo groups, respectively. Based on the annual rate of decline in weight, in the nintedanib group, 397 (62.2%) patients had weight gain or ≤ 5% weight loss and 241 (37.8%) had > 5% weight loss over 52 weeks, and in the placebo group, 338 (79.9%) had weight gain or ≤ 5% weight loss and 85 (20.1%) had > 5% weight loss over 52 weeks. Compared with patients with ≤5% weight loss, patients with > 5% weight loss over 52 weeks had a greater proportion of females, lower mean FVC and DLco % predicted at baseline, and higher (worse) mean SGRQ total score at baseline  was numerically greater in patients with > 5% than ≤5% weight loss over 52 weeks (− 312.7 [SE 32.2] versus − 199.5 SE 14.4] mL/year)   , the difference between nintedanib and placebo was consistent with the original analysis  Figs. ; Table 2In both the nintedanib and placebo groups, the proportions of patients with adverse events of decreased appetite, weight decrease, and progression of IPF were greater in patients with lower baseline BMI and in patients with > 5% than ≤5% weight loss over 52 weeks Tables  and 4. Tpost-hoc analyses of pooled data from the INPULSIS trials suggest that the rate of decline in FVC over 52 weeks was greater in untreated patients with IPF who had lower BMI at baseline, and in patients who had > 5% weight loss during the trials. The effect of nintedanib on reducing the rate of FVC decline was numerically more pronounced in patients with lower BMI at baseline and in patients who had > 5% weight loss during the trials. The rate of decline in FVC in nintedanib-treated patients was similar between the subgroups by baseline BMI and by weight loss during the trials.These Our finding that weight loss was associated with a faster decline in FVC is consistent with a retrospective analysis of two cohorts of patients with IPF (a Japanese cohort of 124 patients and a UK cohort of 86 patients), which found a greater rate of FVC decline in patients with weight loss > 5% than ≤5% over 1 year , 19 and Low BMI and low fat-free mass are common in patients with IPF . Weight Modelling of pharmacokinetic data from patients with IPF treated with nintedanib showed that age, weight, Asian race, smoking, and lactate dehydrogenase levels have small to moderate effects on nintedanib exposure, which are within the range of inter-patient variability and do not warrant a priori dose adjustment , 30. As post-hoc, that the duration of follow-up was only 52 weeks, that the reasons for weight loss could not be determined, that there were differences between the subgroups by BMI and weight loss beyond factors that could be adjusted for, and that there were too few underweight (BMI < 18.5 kg/m2) patients (n = 8) for them to be analysed as a subgroup. While our analyses indicate potential associations between BMI or weight loss and FVC decline, and it is possible that there are links between weight loss and fibrotic processes, we have not demonstrated causal relationships. The limitations of BMI and weight loss as measures of nutritional status and general health must be considered. Patients’ weight loss prior to enrolment in the INPULSIS trials is unknown.Strengths of our analyses include the large sample size (1061 patients with a diagnosis of IPF confirmed by the investigator) and the relatively small amount of missing data on FVC, BMI and weight loss. Our analyses also have several limitations, including that they were These analyses of data from the INPULSIS trials showed that patients with IPF who received placebo and had a lower BMI at baseline, or a weight loss > 5% over 52 weeks, had a greater annual rate of decline in FVC. Nintedanib reduced the rate of decline in FVC both in patients with lower and higher BMI, and in patients with and without weight loss. Active management of the adverse events that may be associated with nintedanib is important to help patients remain on therapy.Additional file 1: Supplemental Appendix 1. Statistical analyses.Additional file 2: Supplemental Appendix 2. List of investigators [1].Additional file 3: Supplemental Table 1. Baseline characteristics in subgroups of patients by BMI  at baseline. Additional file 4: Supplemental Table 2. Outcomes in subgroups of patients by BMI below and at least the median at baseline.Additional file 5: Supplemental Table 3. Annual rate of decline in FVC (mL/year) over 52 weeks in the overall trial population. Additional file 6: Supplemental Table 4. Baseline characteristics in subgroups of patients by weight loss ≤5 and > 5% over 52 weeks . Additional file 7: Supplemental Figure 1. Scatter plots showing the correlation between BMI at baseline and FVC (mL) at baseline (A) and between BMI at baseline and the rate of decline in FVC (mL/year) assessed over 52 weeks (B).Additional file 8: Supplemental Figure 2. Annual rate of decline in FVC (mL/year) over 52 weeks in subgroups of patients by weight loss over 52 weeks (based on the change from baseline in weight at week 52).


Clinical outcomes of novel coronavirus 2019 disease (COVID‐19) in onco‐hematological patients are unknown. When compared to non‐immunocompromised patients, onco‐hematological patients seem to have higher mortality rates.We describe the characteristics and outcomes of a consecutive cohort of 24 onco‐hematological patients with COVID‐19 during the first month of the pandemic. We also describe variations in healthcare resource utilization within our hematology department.P = .01) and D‐dimer ≥900 mcg/L (P = .04). With respect to indirect effects during the COVID‐19 pandemic, and when compared with the prior 3 months of activity, inpatient mortality (excluding patients with COVID‐19 included in the study) increased by 56%. This was associated with a more frequent use of vasoactive drugs (+300%) and advanced respiratory support (+133%) in the hematology ward. In the outpatient setting, there was a reduction in initial visits (−55%) and chemotherapy sessions (−19%). A significant increase in phone visits was reported (+581%).Data from patients between the first month of the pandemic were retrospectively collected. Clinical and logistic data were also collected and compared with the average values from the prior 3 months of activity. Prevalence of COVID‐19 in our hematological population was 0.4%. Baseline characteristics were as follows: male sex: 83%, lymphoid diseases: 46%, median age: 69 (22‐82) years. Median follow‐up in survivors was 14 (9‐28) days and inpatient mortality rate was 46%. Average time to moderate/severe respiratory insufficiency and death were 3 (1‐10) and 10 (3‐18) days, respectively. Only 1 out of every 12 patients who developed moderate to severe respiratory insufficiency recovered. Upon univariate analysis, the following factors were associated with higher mortality: age ≥ 70 years (COVID‐19 pandemic is associated with elevated mortality in hematological patients. Negative indirect effects are also evident within this setting. In the first part of the study, we analyzed the direct effects of COVID‐19 on a consecutive series of onco‐hematological patients. We retrospectively collected clinical data from patients between March 13, 2020 (the first day of government‐imposed restrictions related to COVID‐19 in Spain) and April 12, 2020. Data cut‐off date was April 19, 2020. Data were collected via a retrospective chart review. All patients were ≥18 years old and had an onco‐hematological malignancy. COVID‐19 diagnosis and response were confirmed in accordance with World Health Organization criteria.2/FiO2 < 300 mmHg), and average time from diagnosis to death. Descriptive data were reported as counts and percentages. The proportion of deaths was compared among baseline characteristics using chi‐squared test or Fisher's exact tests depending on the number of events. The following variables were considered: female vs male, age ≥ 70 years, ongoing chemotherapy (defined as last chemotherapy within the last 30 days) vs follow‐up, active disease vs cured disease, 0‐1 line of therapy vs ≥1, presence of comorbidities  vs no comorbidities, D‐dimer ≥900 mcg/L (median of study population) vs <900 mcg/L, radiological evidence of low tract respiratory infection in chest X‐ray vs negative radiological findings. The analysis was performed with STATA statistical software version 13.0.The primary endpoint of the study was to describe the all‐cause in‐hospital mortality rate in patients with hematologic neoplasms and a confirmed diagnosis of COVID19 infection, defined as the proportion of patients with COVID‐19 who died to the overall cohort. Secondary endpoints included: description of patient characteristics, average time from diagnosis to moderate/severe respiratory insufficiency  performed . Also, we described the number of patients without COVID‐19 infection who underwent sub‐intensive care procedures in the hematology ward , as well as the number of patients who required intensive care unit (ICU) admission. For the outpatient setting, the analysis was divided into the following clinical units: HCT, acute leukemia and myelodysplastic syndromes, myeloproliferative diseases, lymphoid malignancies, multiple myeloma and other monoclonal gammopathies, and interventional clinical trials. We reported the following for each unit: the number of total visits, initial visits, in‐person follow‐up visits, phone visits, and daily sessions of chemotherapy/supportive therapy administered in the outpatient clinic.2.2Our institution is a tertiary oncology referral hospital for adult patients, comprising four clinical departments  and two services (Radiology and Pharmacy). Each department has its own ward and outpatient visitor area; however, outpatient therapy space is shared. Furthermore, the hospital has its own infectious disease specialists, and endocrinology and nutrition staff. For all other services, including the intensive care unit, the hospital collaborates with Hospital Universitari de Bellvitge . Due to the COVID‐19 pandemic, our center and activities underwent structural reorganization per regional, national, and international recommendations.Palliative care and the emergency units of the hospital were converted into COVID‐19 wards. In order to prevent nosocomial COVID19 outbreaks in a high‐risk ward, nasopharyngeal swabs for COVID19 were performed in all patients 24 to 48 hours before their scheduled admission. Due to the restrictive triage protocol, no change in room pressure was implemented. No family members were authorized to stay with patients during their hospital stay. Additionally, physicians were separated into two different teams for each ward service  to minimize the loss of workforce due to a possible contagion. Outpatient staff was physically confined to specific areas. Teleworking was encouraged for all activities not requiring physical presence at the hospital. Prior agreement with the ICU to have a referral intensive care specialist in the hematology ward was made. This strategy was implemented with the objective to delay transfers of critical patients and assume more clinically complex cases, defined as those patients who require vasoactive drug support and/or present with rapid acute respiratory insufficiency and need noninvasive ventilation with high‐flow oxygen therapy. Patients admitted to the onco‐hematological ward had the same pharmacological treatments available as those admitted to the ICU, including tocilizumab and high‐dose corticosteroids.33.1P = .01), D‐dimer ≥900 mcg/L (P = .04), and female sex (P = .02). Of the 17 potential patients who developed respiratory insufficiency, eight required advanced respiratory support. One patient required endotracheal intubation and ICU admission; two received non‐invasive mechanical ventilation in the hematology ward and emergency department, respectively. Five patients received high flux nasal cannula in the ward.Taking into consideration patients followed‐up in the last 2 years in our hematology department (n = 6779), the expected prevalence of COVID‐19 in our oncological population was 0.4%. This figure is similar to that reported in the general population of the same geographic area (0.5%).3.2During the first month of pandemic restrictions, monthly inpatient activity of non‐COVID‐19 patients was affected Table . The totOutpatient activity was severely affected during the COVID‐19 period. The total number of first visits dropped by 55% Figure . In addi4We report for the first time a descriptive analysis of the direct and indirect effects of the COVID‐19 pandemic on a tertiary oncology referral center. Firstly, we performed an analysis of the characteristics of symptomatic COVID‐19 from a consecutive cohort of hematological patients. The prevalence of symptomatic COVID‐19 in our patients' population was comparable to that of the general population. This is different from what was previously reported in a Chinese study, which suggested a higher risk of contagion in the oncological population.Several factors could explain this difference. First of all, cancer is generally a disease of the elderly. Older age was associated with increased COVID‐19‐related mortality in almost all epidemiological studies. Considering that the median age of our study population was 69 years, this could have likely contributed to poor survival.The use of different types of therapy could also influence this outcome. For example, B‐cell depletion by use of rituximab could potentially interfere with an adequate antibody response to the SARS‐CoV‐2. The use of checkpoint inhibitors capable of eliciting an increased immune response could have a role in favoring acute respiratory distress syndrome within this setting. More data are needed to clarify this hypothesis. In addition, being neutropenic could favor bacterial super‐infections during COVID‐19.Nonetheless, such a high mortality rate appears superior to that of any other common seasonal respiratory virus , which usually does not exceed 10% to 20%, including in severely immunocompromised patients.We believe that the high mortality rate cannot be attributed exclusively to the COVID19, especially considering that 20/24 patients (83%) had an active underlying hematological neoplasm. However, in all patients who died, the leading cause of death was respiratory insufficiency, which makes us believe that COVID‐19 had a critical role on the poor patients' outcomes.Our study also confirms elevated age and D‐dimer levels as risk factors associated with higher mortality.Finally, we suggest that onco‐hematological patients could deteriorate faster than the general population. This is true in terms of time from diagnosis to respiratory deterioration and death. A similar observation was reported elsewhere.see Table Considering the indirect effects of COVID‐19, it is important to describe how such a pandemic was able to cause the collapse of a modern healthcare system. Following national and international recommendations, we reduced our daily activity and reserved it only for selected necessary cases. At an inpatient level, we observed an increased mortality in our patients who did not present with COVID‐19 infection. This could probably be explained by the need to attend to more palliative care patients in the hematology ward during the COVID‐19 period. To a lesser extent, a limited use of ICU and other essential hospital services could have played a role. Increased COVID‐19 mortality is in line with data from the national registry, which reported a significant increase in death from all causes during March 2020. As it concerns outpatient care, the number of first visits decreased significantly in all units . Larger studies with a prolonged follow‐up will be necessary to understand in‐depth clinical characteristics of this new infection and its indirect effects. A more comprehensive view of how a pandemic could affect a hematological center in the modern era could facilitate the conception of improved contingency plans in the advent of health emergencies.The authors have declared no relevant conflicts of interest related to this study.Conceptualization, M.C., A.M., C.M.; Methodology, A.M.; Investigation, M.C., A.M, C.M., R.P., E.G.B., M.A., A.A.P., H.P., M.Q.S., I.C., M.P., V.C., C.B., A.C.O., G.S.L., G.M.G., S.M., C.B., C.G., E.D.D.; Formal Analysis, M.C., A.M., C.M.; Writing—Original Draft, M.C., A.M., C.M., R.P., E.G.B., M.A., A.A.P., M.Q.S., G.M.G., E.D.D., A.S.; Writing—Review & Editing, M.C., A.M., C.M., A.S.; Visualization, M.C., A.S.; Supervision, A.S.; Project Administration: A.M.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The Institutional Review Board (IRBs) of the Catalan Institute of Oncology‐Durán i Reynals Hospital approved this study and patients provided informed consent for data collection.


Bombus terrestris. In B. terrestris, female larvae experience a queen‐dependent period during which their caste fate as adults is determined followed by a nutrition‐sensitive period also potentially affecting caste fate but for which the evidence is weaker. We used mRNA‐seq and qRT‐PCR validation to isolate genes differentially expressed between each caste pathway in larvae at developmental stages before and after each of these periods. We show that differences in gene expression between caste pathways are small in totipotent larvae, then peak after the queen‐dependent period. Relatively few novel  genes were differentially expressed between castes, though novel genes were significantly enriched in late‐instar larvae in the worker pathway. We compared sets of caste‐associated genes in B. terrestris with those reported from the advanced eusocial honeybee, Apis mellifera, and found significant but relatively low levels of overlap of gene lists between the two species. These results suggest both the existence of low numbers of shared toolkit genes and substantial divergence in caste‐associated genes between Bombus and the advanced eusocial Apis since their last common eusocial ancestor.The queen‐worker caste system of eusocial insects represents a prime example of developmental polyphenism  and is intrinsic to the evolution of advanced eusociality. However, the comparative molecular basis of larval caste determination and subsequent differentiation in the eusocial Hymenoptera remains poorly known. To address this issue within bees, we profiled caste‐associated gene expression in female larvae of the intermediately eusocial bumblebee  In these, multiple phenotypes can develop from a single, species‐specific genome via differing environmental inputs Kapheim, . UnderstCaste determination in eusocial Hymenoptera represents the branching point when diploid females cease being totipotent  and their caste fate becomes fixed. Caste differentiation describes the developmental processes occurring along each caste‐specific pathway. In species in which caste determination occurs in larvae, relatively few studies have profiled gene expression changes across sensitive periods of caste determination. To do so is important because genes isolated from such studies are likely to play key roles in the genetic pathways underpinning eusocial castes.Genes associated with caste determination and/or differentiation in eusocial species have been hypothesised to fall into two broad categories: (a) Novel genes, i.e., taxonomically‐restricted genes that confer the convergent traits associated with eusociality in each independent eusocial lineage  associated with caste differentiation that was expressed from the intron of a protein‐coding gene, vitellogenin‐like, suggesting that Bte‐miR‐6001 and vitellogenin‐like could have a role in caste differentiation in this species.Previous studies of differential gene expression in B. terrestris at developmental stages straddling the two sensitive periods. Using mRNA‐seq, we profiled larvae before the queen‐dependent period (first/second‐instar larvae), between the queen‐dependent period and nutrition‐sensitive period (third‐instar larvae), and after the nutrition‐sensitive period (fourth‐instar larvae). We term these “early‐”, “mid‐”, and “late‐instar” larval stages, respectively. In general, we refer to genes isolated by these procedures as genes potentially affecting caste determination and/or differentiation. However, we note that for the reasons described above, genes differentially expressed between caste pathways in late‐instar larvae are most likely to be associated with caste differentiation, whereas genes most likely to affect caste determination are those differentially expressed during the queen‐dependent period, i.e., immediately before the mid‐instar larvae were collected. We used gene ontology (GO) analyses and investigations of individual genes and gene families to identify key caste‐associated processes and to select the subset of genes differentially expressed in the mRNA‐seq data that would be most likely to affect caste determination and/or differentiation. We then validated expression differences in these genes using qRT‐PCR. Within our first aim, we tested three interrelated hypotheses regarding caste‐associated gene expression in B. terrestris. Hypothesis 1 was that differential gene expression between queen‐ and worker‐destined larvae is absent in early‐instar larvae (as they are totipotent), greater in mid‐instar larvae (as they have embarked on one of the caste pathways) and greatest in late‐instar larvae (as they show the greatest phenotypic differences). Hypothesis 2 provided a test of the novel genes hypothesis, which predicts that a relatively high proportion of caste‐associated genes are novel, and that novel and unannotated genes are over‐represented in the worker pathway  and vitellogenin‐like are upregulated in the queen pathway. This was because the gene Dnmt3 is an essential component of methylation and previous studies have suggested roles for methylation in queen determination and/or differentiation in A. mellifera and in the control of reproduction in B. terrestris workers  to profile the gene expression differences associated with the sensitive periods of larval caste determination in B. terrestris, and to compare such differences in larval caste pathways across separate eusocial taxa differing in their degree of eusociality (Bombus and Apis).Our second aim was to compare expression data from queen‐ and worker‐destined larvae in 22.12.1.1B. terrestris. (Detailed methods are available in We obtained samples of queen‐destined larvae (by removing colony queens from a subset of colonies) and worker‐destined larvae (by retaining colony queens in a subset of colonies) of 2.1.2We extracted total RNA following methods detailed in the 2.1.3https://github.com/sRNAworkbenchuea/UEA_sRNA_Workbench) to quality filter mRNA‐seq reads and aligned them to the B. terrestris reference genome  as a significantly differentially expressed gene (DEG) between caste phenotypes when the maximal confidence intervals  for queen‐ and worker‐destined larvae in a given developmental stage did not overlap  between mRNA‐seq data analysed using the PatMan/confidence‐interval pipeline and qRT‐PCR data  when OFC >1 between caste phenotypes within a larval stage and after accounting for multiple unannotated exons making up a longer multiple‐exon transcript ; (b) unannotated but not‐novel genes ; (c) unannotated, novel non‐coding RNA ; and (d) unannotated, novel coding RNA . We used BLASTX/BLASTN to compare each transcript with the NCBI nucleotide and protein databases, defining each transcript as novel if either search returned a hit to the genus Bombus but no other genus at E value ≤ 1 × 10−5 , we classified transcripts that mapped to the genome and were found to be HDEGs into four annotation classes: (a) Annotated genes , against a background set of D. melanogaster orthologues with B. terrestris  BLAST search  Showed the highest degree of differential expression in each of the phenotypes; (b) was classed as a HDEG  within at least one of the three developmental phenotypes; (c) was classed as a HDEG within at least two of the three developmental phenotypes; (d) fell within a gene family with at least one other member that was also highly significantly differentially expressed between caste phenotypes within at least one of the three larval stages; and (e) had been linked by published studies to caste determination and/or differentiation in ra Table . In totara Table .Dnmt3 and vitellogenin‐like upregulation in queen‐destined larvae), we inspected the DEG lists for evidence of upregulation. We found that these two genes were not among the DEGs in the mRNA‐seq data. However, as these could have been false negatives, we added these two genes to the 14 genes already selected for qRT‐PCR. Hence, in total, we assayed gene expression in 16 target genes using qRT‐PCR  between queen‐ and worker‐destined larvae . We then assessed whether for each comparison the relatively quantified Cq values obtained from qRT‐PCR showed the same results as the mRNA‐seq data (see 2.22.2.1B. terrestris and A. mellifera), we used a RBH BLAST analysis . We then compared these B. terrestris orthologue lists with gene lists from two studies of A. mellifera: (a) Cameron et al.  Align, and conduct differential expression analysis on, the three raw mRNA‐seq data sets from the current study, Cameron et al.  conduct the HISAT2/HTSeq comparative analysis in (a) but with the diapause‐associated genes excluded  Technical differences in the analysis pipelines used across different studies; or (b) biological differences occurring from queen diapause in n et al.  and He en et al. , using t, et al.  and test33.13.1.1We sequenced 742,242,464 reads  across all 17 retained libraries Table . A total3.1.2Plrp2‐like; XM_003398387.3) in MQ being the most differentiated gene in mid‐instar larvae with an OFC of 5.8, followed by P17/29C‐like protein DDB_G0287399  in MW with an OFC of 3.94 , lending partial support to Hypothesis 2 (novel genes hypothesis). Worker‐destined larvae had proportionally more novel HDEGs (with 14/92 (15.2%) and 7/14 (50.0%) of the total upregulated transcripts being novel in MW and LW, respectively; Figure p = 0.042 and p = 0.813, respectively). Of the 33 novel transcripts, one  was predicted to be protein‐coding and all remaining ones were predicted to be long non‐coding RNAs (lncRNAs)  were annotated transcripts, 79/200 (39.5%) were unannotated transcripts and 26/200 (13%) were uncharacterised transcripts Figure . Of the s) Table .3.1.4BLAST search between the transcriptome of B. terrestris and the annotated gene set of D. melanogaster revealed 7,689 orthologous genes . For each of the remaining 6/17 HDEG comparisons that were not significantly differentially expressed in the same direction in the qRT‐PCR data, the mean expression value in these data was higher in the same phenotype as in the mRNA‐seq data. Therefore, the trend in differential expression for these six genes was the same for both methods (Table The selected genes of interest that were HDEGs fell into five gene categories (described in ds Table . In addids Table .Dnmt3 and vitellogenin‐like upregulation in queen‐destined larvae), as neither gene was differentially expressed between caste pathways in either the mRNA‐seq or the qRT‐PCR data , we isolated 7,598 orthologues that were present in the mRNA‐seq data  from B. terrestris (current study) and in the microarray data from A. mellifera in Cameron et al.  in A. mellifera  in A. mellifera  and mRNA‐seq data from the second focal A. mellifera study  and a total of 14 HDEOs that showed significant upregulation in the same direction in the current study and at least one of the A. mellifera comparison studies (Table Hexamerin (XM_003401733.3), Krüppel‐homologue 1 , and probable CYP303a1 (XM_003396659.3). They all showed upregulation in queen‐destined larvae in the mRNA‐seq and qRT‐PCR data from B. terrestris in the current study and in the data from at least one A. mellifera study, which supports a caste‐associated role for these genes in both lineages. However, relatively few HDEOs showed similar expression patterns in both species (a maximum of 14/54), and the significant overlaps between the current study and the A. mellifera studies involved low numbers of genes , greater in mid‐instar larvae (as they have embarked on one of the two caste pathways) and greatest in late‐instar larvae  were novel. In addition, novel genes were significantly over‐represented in the genes upregulated in LW , of which 7/14 (50%) genes were novel compared to 5/40 (12.5%) genes upregulated in LQ being novel. These results partially support Hypothesis 2 as a proportion of caste‐associated genes were novel, and the worker pathway was relatively enriched for novel genes. However, studies of other eusocial insects have found up to 75% novel transcripts among caste‐associated genes and a percentage of novel genes among genes upregulated in workers of up to 81.5%  was predicted to be protein‐coding (feature ID: NW_003568470.1_7_877) and the rest were predicted to be lncRNAs. The large proportion of novel lncRNAs, together with previous results isolating caste‐associated miRNAs in larvae in B. terrestris  genes would be associated with caste determination and/or differentiation, and that novel genes would be over‐represented in the worker pathway genes (see Introduction). We found that, of the 105 unannotated/uncharacterised HDEGs in Bombus and Apis, as in Apis metabolism genes are generally more closely associated with the queen pathway , while worker‐destined larvae tended to be enriched for genes involved in metabolism Figure . This diy Figure .Hexamerin, Kr‐h1, Takeout‐like, and relatives of these genes) have previously been shown to be up‐ or downregulated in response to JH in either B. terrestris or other eusocial insects, and these genes were upregulated in queen‐destined larvae in the mRNA‐seq data from the current study  is consistent with this role, as the mid‐instar stage follows the queen‐dependent period, suggesting that these genes, and their possible interactions with JH, are potentially important candidates for involvement in caste determination and/or differentiation. Takeout‐like (XM_003397243.2) also has a negative relationship with JH in brain of adult A. mellifera workers  among caste‐associated genes in B. terrestris, and two of the CYP genes in this study  have been shown to be essential for proper development and metamorphosis in other insects  genes and Takeout‐like) that were upregulated in queen‐destined larvae in B. terrestris have been linked to sex differentiation in other insects  upregulated in late‐instar queen‐destined larvae  maintain Bte‐miR‐6001 at a lower level in late worker‐destined larvae than its host gene.Hypothesis 3 predicted that two genes, a Figure . For Dnm4.2B. terrestris data set from the current study with two published A. mellifera data sets  applied across all three data sets . However, we found that: (a) Diapause‐associated genes were not significantly over‐represented among the lists of caste‐associated genes in B. terrestris ed Table . TherefoHexamerin, Kr‐h1, and CYP303a1 (XM_003396659.3); Table A. mellifera larvae maintain higher overall levels of JH than worker‐destined larvae (Wirtze, B. terrestris, topical application of JH to worker‐destined larvae causes them to have queen‐like characteristics as adults (Wirtze & Beetsma, Bombus and Apis, significant but low overlaps between caste‐associated genes in related eusocial lineages (Berens et al., Bombus and Apis since their last common eusocial ancestor (cf. Linksvayer & Johnson, As discussed above, some of the genes that overlapped between the two species (e.g.,  Wirtze,  and, as D.H.C., T.D., and A.F.G.B. designed the research. D.H.C., M.L., M.S., and D.C.P. performed research. D.H.C., A.W., D.C.P., and I.M. analysed data. D.H.C. and A.F.G.B. wrote the manuscript. All authors provided comments on the manuscript.Supplementary information including Figure S1‐S6, S9‐S14Click here for additional data file.Figure S7Click here for additional data file.Figure S8Click here for additional data file.Table S1‐S9, S11‐S18Click here for additional data file.Table S10Click here for additional data file.