Effects of Llight and temperature on vertical migration of benthic diatoms

 



 

 


AbstractVertical migration of two benthic[JH1]  diatoms was studied inas a response to different light intensityy and temperature[JH2] . Using An[JH3]  Imaging-PAM (P pulse-amplitude-modulated) fluorometer (Imaging-PAM), and [JH4] minimal fluorescence (F0) waswere used to monitor diatom biomass variation in surface sediments, and rapid light curves (RLCs) were usedapplied to assess the photosynthetic activities of the tested diatoms. Both Cylindrotheca closterium and Nitzschia sp.[JH5]  presented increasing motility under higher-temperature conditions. However, C. closterium, with its long valves, hadshowed more than twice the migratory speed asof the shorter-valved Nitzschia sp.[JH6]  with shorter valves. The Ttwo light intensities, of 100 and 250 µmol photon[JH7]  m-2 s-1, had different effects on C. closterium, but had not effects on Nitzschia sp. Consequently, there was no light/temperature interaction between light and temperatureeffect on the vertical migration of Nitzschia sp. With lower photosynthetic capacity and smaller cell size, Nitzschia sp., with its low photosynthetic capacity and smaller cell size, responded differently from C. closterium. Although withthe mechanism is[JH8]  complicated, mechanism, light and temperature were proved to greatly influence the migratory behavior of the benthic diatoms, and inducethrough species-specific response of different diatom speciesinducements.

Keywords-: benthic diatom; chlorophyll fluorescence; light; temperature; vertical migration

                                                                                                                                                               I.           Introduction

Benthic diatoms inhabiting intertidal sediments exhibit vertical migratory rhythms within the upper sediment layers, which are associated with diurnal and tidal cycles [1, 2, 3, 4].[JH9]  Typically, when large numbers of diatoms aggregate inon thea sediment surface, they forming a biofilm, which, due to the fucoxanthin pigment, exhibits a golden brown color is easilyreadily observed withapparent to the naked eye due to the fucoxanthin pigment of diatoms [5, 6]. Since vVertical migration of benthic diatoms, hashaving been largelywidely recognized as a key controlling factor ofdetermining[JH10]  short-term variability in benthic primary productivity, it has been studiedreceived increasingly research attention in recent years [7, 8, 9].

Light, As a mainprincipal[JH11]  triggering factor for benthic diatoms’ vertical migration, light  controls the migration rhythms[JH12]  [4, 10, 11], and also affects the photosynthetic activity of benthic diatoms [12, 13, 14]. Although And though the mechanism isis complicated, under laboratory conditions the temperature does influence the migration rhythmtemperature has also been found to influence the migration rhythm of field samples under laboratory conditions [15, 16], as well as the photosynthesis of field-sample benthic diatoms [17, 18].

In surface sediments onthe  intertidal flats, the light intensity and temperature haveshow major spatial and temporal gradients in surface sediment,that varying alongwith the rise and recession of with the tides rising and receding [19]. The lLight intensity couldcan change from very high (exceeding 2000 µmol photon m-2 s-1) at low tide and solar noon[JH13]  to littlevery low or nothing at high tide[JH14]  [JH15] light reaches the sediment surface[JH16]  during low tide at solar noon [14, 20]. The sSurface temperature has been reported that hadexhibited a maximum daily change up toas large as 18 oC, atfor a rate of 3 oC h-1 [12],; and in temperate regions, temperature in the upper 200 µm of muddy sediments can easily changeshift by 10 oC during emersion periods, withat rates ofas high as 4 oC h-1 [21].

Responding to varying environmental factors,The migratory ability of benthic diatoms in response to varying light, temperature and other factors enables their success within the sedimentary environment. It has been observed in situ that the diatoms concentrated at a depth of 1 mm can migrate up[JH17]  to the surface in 1.5 hours[JH18]  in situ [22]. And thethis migration has been found isto be species-specific, viz.that is, species surfacings altereddiffered at differentaccording to the time during aof day [9, 16, 23]. [JH19] 

Fluorescence techniques usingutilizing thean imaging pulse-amplitude-modulated fluorometer (Imaging-PAM) hashave been widely used onapplied to the measurement of microphytobenthic biomass and photosynthetic activity [14, 24, 25, 26, 27]. ThePAM-measured minimum fluorescence (F0) measured by PAM is proved to have a linear relationship with microphytobenthic biomass [24, 28, 29, 30]. And Aamong the various PAMs, Imaging-PAM not only allows the quick, non-intrusive and sensitive measurement as others[JH20] , but also hasoffers the uniquely advantageouss utility of measuring larger areas and numbers of interesting points simultaneously onin one image. Using Imaging-PAM, then, can decreaseminimize experimental error which caused byresulting from the prolonged time required for measuring samples individually.

Until now, no study has investigated the two-factor effects of light and temperature on the vertical migration of benthic diatoms. In thisthe present study, using Imaging-PAM and a miniaturized setup, we aimed to investigated the vertical migration of two diatom species in response to different temperature and light conditions, and analyzed their migration mechanisms both photo-[JH21] physiologically and morphologically, in order to assistenhance further the synthetic understanding of the microphytobenthos (MPB) ecology in in situtheir[JH22]  environments.

                                                                                                                                            II.         Materials and Methods

Unialgal cultures of Cylindrotheca closterium (hereafter C. closterium) and Nitzschia sp. (supplied by the Korea Marine Microalgae Culture Center, Busan, South Korea) were grown in 2 L flasks[JH23]  with f/2 medium [31] withand pre-filtered seawater (0.2 µm membrane filter; salinity,: 34 psu). All[JH24]  of the cultures were incubated under constant conditions atincluding  20 oC temperature withand a 12 h: 12 h light (100 µmol photon m-2 s-1): dark photoperiod. of 12 h: 12 h. The growth rates were monitored through increases in the chlorophyll a concentration and cell number. Once the cultures reached the stationary phase, diatom cells were deposited homogenously[JH25]  on glass microfiber filters (porosity: 2.3 µm) by slow filtration (< 0.1 MPa), forming artificial biofilms. The Ccell sizes of  C. closterium and Nitzschia diatomssp. waswere measured under a light microscopy using an AxioVision LE (Allied High Tech Products, Inc.,; Rancho Dominguez, California, USA)., and The sizesfound to be (mean ± SD) of C. closterium and Nitzschia sp. were 103 ± 6.3 × 4.1 ± 0.3 µm and 18 ± 2. 1 × 2.1 ± 0.2 µm, respectively.

Prior to the experiment, the samples in 24-well plates were prepared from the adjusteding sample height with 3.0 g treated sediment (removed of all organic materials). in a well[JH26] . Small circulare [JH27] biofilms were cut from the artificial biofilms into the same size of the inner diameter of the wells, and then were putplaced on the top of the 3.0 g sediment base. The Iinitial minimum fluorescence (F0)[JH28]  was measured by Imaging-PAM after a 5 min dark-adaptation period. Rapid light curves (RLCs) were useddrawn forto assessing the photosynthetic activities, wherethe samples werehaving been exposed to twelve incremental steps of irradiance (10s per step) ranging from 0 to 701 µmol photon m-2 s-1. Relative electron transport rates (rETR) were givenobtained byfrom the RLCs. measurement[JH29] . Three Pphotosynthetic parameters, including the maximum rETR (rETRmax), the minimum saturating irradiance (Ekk) and the Llight utilization coefficient (α), were derived from thean rETR light response curve fitted to the model of Platt et al.’s model [32]. After measuring the F0 and RLCs, these circularle biofilms were covered with a sediment layer approximately 2 mm thick sediments (125–250 µm). All of the wells with samples were maintained saturated with f/2 medium duringthroughout the experiment. process with f/2 medium.

The well plate with samples was put on a fixed mounting stand position under the measuring head of the Imaging-PAM (Max/L, Walz, Germany). Before measuring the fluorescence, areas of interest (AOIs) of circular-sample size were defined underin Live Video Mode,. with the same size of circle samples. The same AOIs were used consistently used duringover the course of the whole experimentation. The fFluorescence was induced by royal blue (450 nm) 3 W Luxeon LEDs, which have standard intensity of 0.5 µmol m-2 s-1 standard intensity and 1 ~ 8 Hz modulation frequency. between 1 and 8 Hz.

The experiment of the light and temperature effects on the two species’ vertical migrations was carried on two specieswere studied under two light intensityies[JH30]  (100 and 250 µmol photon m-2 s-1) and three temperature (10, 20, 30 oC) conditions, viz. incubated the prepared well-plate samples were incubated under 10 oC-100 µmol photon m-2 s-1, 20 oC-100 µmol photon m-2 s-1, 30 oC-100 µmol photon m-2 s-1, 10 oC-250 µmol photon m-2 s-1, 20 oC-250 µmol photon m-2 s-1, and 30 oC-250 µmol photon m-2 s-1, environments, representing a totally of six treatments for each species. Each treatment had four replicates. The F0 was measured by Imaging-PAM, after a 5 min dark adaptation, at 0 h, 1 h, 2 h, 3.5 h, 5.5 h and 7 h. The sediment surface F0 of each sample was usedreferenced to monitordetermine[JH31]  the surface biomass increase due to the diatoms migrating upmigration from the circulare biofilm to the sediment surface.

To compare the effects of the different incubation conditions on the two species (i.e. to determine the significant differences), a univariate analysis of variance, followed by post-hoc Tukey tests waswere carried out to test significant difference using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

                                                                                                                                                                        III.        Results

The initial F0, values, which as measured on the small circulare biofilms before the latter were covereding with sediment, were 0.0440 ± 0.0026, 0.0422 ± 0.0021 (arbitrary units) for  C. closterium and Nitzschia sp., respectively. The Tthree photosynthetic parameters (Ek, α and (rETRmax, Ek and α)[JH32]  of C. closterium arewere all higher than those of Nitzschia sp. (Table I). The migratory responses of the two species to the different light intensities and temperatures are illustrated in Fig.1. After 2 h incubation, for 2 hours, diatoms cells of the two species began migrate up to the sediment surface. From 2 to 7 h incubation, moreadditional[JH33]  diatoms cells of the two species migrated, up and presentinged a different migratory response to the individual[JH34]  conditions. Generally, the diatom cells migrated quickerfaster at higher temperatures than at lower temperatureones, with sequence ofin the order 30 oC > 20 oC > 10 oC for both species. And C. closterium showed a higher motility by almost twice F0 as that ofthan did Nitzschia sp., sp.almost twice its F0[JH35] , after 3.5 h incubation.

Comparing the effects of the six conditions on the two species afterfor certainthe several[JH36]  incubation timetimes, two species showedthere was no significant (P > 0.05) difference at 2  h incubation, but there were extremely significant (P < 0.001) differences at 3.5 h, 5.5 h and 7 h incubation. under the same light and temperature conditions[JH37] . With respect to temperature, the two species responded significantly (0.01 < P < 0.05) differently forbetween 2 h toand 7 h incubation. There was no statistically significant difference between two speciesthem in response to the two light intensities at 2 h, but they differed significantly at 3.5 h, very significantly (0.001 < P < 0.01) at 5.5 h and extremely significantly at 7 h incubation (Table II). Generally, the differences between individual conditions increased along with incubation time.

For C. closterium, the light intensity showed no statistically significant effects on upward migration at 2 h incubation, but haddid so significantly difference at 3.5 h and extremely significantly difference at 5.5 h and 7 h incubation. However, it was obvious that at 10 oC of 3.5 h, 20 oC of 3.5 h and 7 h, the F0 values showed no significant difference between 100 and 250 µmol photon m-2 s-1. Visually, the 100 µmol photon m-2 s-1 light intensity induced more migration than did 250 µmol photon m-2 s-1 for C. closterium at 10 and 30 oC. The temperature effects on the vertical migration of C. closterium were significant from 2 h to 7 h incubation. A Ssignificant or extremely significant difference existedwas evident in each temperature pair comparison between temperatures for 2 h toand 7 h incubation. The interaction between light intensity and temperature for C. closterium was not significant at 2 h incubation, but was significant at 3.5 h, 5.5 h and 7 h incubation (Table II).

TABLE I.            Photosynthetic Parameters of Two Species

Species

rETRmax

(relative units)

α

(relative units)

Ek

(µmol e m-2 s-1)

Cylindrotheca closterium

41.74

0.28

149

Nitzschia sp.

36.11

0.25

147

Figure 1.    Text Box:  Effect[JH38] s[JH39]  of light intensity and temperature on the two species’ upward migration of two species with incubation time.

For Nitzschia sp., the light intensity had no statistically[JH40]  different effect from 2 h to 7 h incubation. The temperature effects on its vertical migration, of Nitzschia sp.though, were significant from 2 h to 7 h incubation. WithIn respect ofof each pair comparison betweenof temperatures, the post-hoc Tukey tests showed no difference between 10 and 30 oC, or 20 and 30 oC at 2 h incubation, as well asor indeed between 20 and 30 oC at 3.5 h incubation,; however, showedthere were manifestly  significant, or very significant, or even extremely significant differences between the temperatures at the other incubation times. There was no interaction between light intensity and temperature for Nitzschia sp. under the designed experimental conditions (Table II).

                                                                                                                                                                   IV.        Discussion

A.       Effects of light and temperature

Lower light intensity usually induces morelarger benthic diatoms migrationsg up to the sediment surface. On intact biofilms of estuarine sediments, Serôdio et al. [25] reported that, on intact biofilms of estuarine sediment, the surface biomass increases under irradiances below 100 µmol photon m-2 s-1 and reaches maximum values under 100-250 µmol photon m-2 s-1, but decreases gradually under higher irradiances as (1000-1500 µmol photon m-2 s-1). In our previous study, we found thethat

TABLE II.          Significant Values offrom Univariate Analysis on Two Species Incubated Under 2 Lights and 3 Temperatures Conditions

 Species

Source

2h

3.5h

5.5h

7h

Cylindrotheca closterium and

 Nitzschia sp.

Temp

0.000

0.000

0.000

0.000

Light

NS

NS

0.001

0.001

Species

NS

0.000

0.000

0.000

Temp * Light

NS

0.048

NS

0.011

Temp * Species

0.000

0.000

0.000

0.000

Light * Species

NS

0.049

0.001

0.000

Cylindrotheca closterium

Temp

0.000

0.000

0.000

0.000

Light

NS

0.038

0.000

0.000

Temp * Light

NS

0.049

0.034

0.002

10*20

0.017

0.000

0.000

0.000

10*30

0.000

0.000

0.000

0.000

20*30

0.000

0.000

0.000

0.000

Nitzschia sp.

Temp

0.025

0.001

0.000

0.000

Light

NS

NS

NS

NS

Temp * Light

NS

NS

NS

NS

10*20

0.022

0.045

0.006

0.004

10*30

NS

0.000

0.000

0.000

20*30

NS

NS

0.020

0.000

NS: Not-significant

concentrated diatoms migrated up to the sediment surface under 50-500 µmol m-2 s-1, but migrated down into deeper sediment after 4 h of illumination under 500 µmol photon m-2 s-1, and showed no obviously migratory behavior under 1000 µmol photon m-2 s-1 [33]. In thisthe present study, we usedapplied 100 and 250 µmol photon m-2 s-1, which could causeinduced maximum upward migration, and and haveto a 2.5 times disparity. AndThat is, the tested species, C.ylindrotheca closterium, presented more upward migration under 100 µmol photon m-2 s-1 than under 250 µmol photon m-2 s-1.

The effect of temperature on the migration is fewrarely studied with respect to motility. Generally, higher but not extreme temperatures could increase the motility of unicellular organism [34, 35]. In thisthe study here concerned, both species showed higher upward migration at higher temperatures, which also recorded inresults consistent with the  research of Cohn et al. [36]. However, lLow temperature (2 oC), by contrast, was reported thathas been found to markedly reduce the migration rhythm of diatoms [15]. Similarly, in our recent field study on temporal variation in the vertical distribution of microphytobenthosMPB, the migration rhythm also was changed, byspecifically when the diatoms[JH41]  stayingremained longer in surface sediment at 5.5~6.6 oC (the daily average temperature) in winter [37]. The Llower motility at lower temperatures could contribute to this phenomenon.

Although the interaction of light and temperature was found only found in C. closterium in thisthe present experimentation, it does not indicatefollow that interactions do not exist in Nitzschia sp. or other species. A prime reason is that temperature is a very important factor influencing almost all physiological activities, including the photosynthetic activity and motility [16, 17, 18, 38]. Adding moreadditional sediment levelslayers could help elucidate the interaction but would also increase the experimental complexity. of experiment. Therefore, one factor effect on more species is suggested to be carried in advance.[JH42] 

 

B.       Species-specific response and motility

In this study,The two species studied here exhibited different motility responses and motility to[JH43]  light and temperature. The Mmotility of C. closterium was about twice as high as that of Nitzschia sp. As already mentioned, Nthere were no effects of light andor of the interaction of light and temperature were found onon Nitzschia sp., which was also different from those ofcontrastingly to C. closterium. In one of our previous studiesy, the species-specific difference also presented itself onin the effects of the light and grain size on the vertical migration for Amphora coffeaeformis and C. closterium [33]. The results of ourthe present[JH44]  study corroborate again the field findings of the species-specific variation in migration [9, 16, 23].

Concerning these various migratory responses, one main origin is the photo-physiological characteristics of different diatoms species. The motive of thebehind upward migration is forthe satisfactionying of their light requirement of their photosynthesis. AndCorrespondingly, species with lower Ek acclimate themselves to lower light intensity. It has been found that Pleurosigma angulatum, which is dominant in diatom biofilms at midday, hasd a higher Ek (between 500 and 600 µmol m-2 s-1), while Nitzschia dubia, which displayedreflecting its rapid vertical migration away from the surface under increasing irradiance, hasd a lower Ek of 300 µmol m-2 s-1 [15]. In thisthe present studyinvestigation, the lower Ek, α and rETRmax indicated that Nitzschia sp. hasd a lower photosynthetic capacity and activity, and that alsoit prefers a lower light intensity than that favored by C. closterium. Therefore,Hence our finding that lessfewer cells of Nitzschia sp. cells migrated up to the sediment surface. [JH45] 

Morphological characteristics could also partly explain the difference ofin the migratory responses, especially the motility difference. Comparing their cell sizes, the C. closterium has long valves (approx. 103 × 4.1 µm or so), but Nitzschia sp. has shorter and thinner valves (approx. 18 × 2.1 µm or so). Small size has been showned to be disadvantageous for cells locomotion through sediment, because this motion also needrequires a substratum to adherewhich by using their extracellular polymeric substances (EPS) can adhere [39, 40, 41, 42]. Small species such as Navicula species hashave been observed using the trails of larger diatoms Pleurosigma angulatum for upward migration [43]. Furthermore, with long and only lightly or partially silicified valves, the cells of C. closterium couldcan move more quicklyer through the sediment aiding by rotating their frustules. Therefore,Thus, the motility of C. closterium could be more than twice as that of Nitzschia sp. in this study. With regard to the different responses to light intensity, the more efficiently light harvesting of a small cell due to the smaller packaging effect [44] could be a reason for whyfor the disparity offact that 100 and 250 µmol photon m-2 s-1 hadve no effect on small species as Nitzschia sp[JH46] .

Another morphological index, the SA/V ratios (i.e. the ratio of surface area to volume), also could also help to elucidate[JH47]  the lesser upward migration of Nitzschia sp. under higher temperatures. In the study ofby Yun et al. [38], the photosynthetic activities of smaller species with larger SA/V (ratio of surface area to volume)[JH48]  ratios were more negatively affected by higher temperature than larger species with smaller SA/V ratios. And they measuredIndeed, the SA/V ratios of Nitzschia sp., in Yun et al.’s measurement, was 2.88 ± 0.08 µm-1, which is more than twice than that of C. closterium (about 1.3µm-1)[1].

 

Acknowledgment

This research was supported by the Korean Ministry of Land, Transport and Maritime Affairs as “Greenhouse Gas Emission Reduction using Seaweeds.” We especially appreciate the financial supported byof “the Fundamental Research Funds for the Central Universities” of the Ministry of Education of China, towhich aided the completione of this manuscript.

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[35]  S.A. Cohn, J.F. Farrell, J.D.Munro, R.L.Ragland, R.E. Weitzell and B.L. Wibisone, “The effect of temperature and mixed species composition on diatom motility and adhesion,” Diatom Res., vol. 18, pp. 225–243, 2003.

[36]  G.y. Du, M. Son, S. An and I.K. Chung, “Temporal variation in vertical distribution of microphytobenthos in intertidal sand flats on Nakdong River estuary, Korea,” Estuar. Coast. Shelf Sci., vol. 86, pp. 62-70, 2010.

[37]  M.S. Yun, S.H. Lee and I.K.Chung, “Photosynthetic activity of benthic diatoms in response to different temperatures,” J. Appl. Phycol., vol. 22, pp. 559-562, 2010.

[38]  L.A. Edger and J.D. Pidkett-Heaps, “Diatom locomotion,” in Progress in phycological Research, F.E. Round and D.J. Chapman, Eds, vol.3, pp. 47-88. Biopress Limited, Bristol., 1984.

[39]  L.A. Edger and M. Zavortink, “The mechanism of diatom locomotion. 2. Identification of actin,” Proceedings of the Royal Society of London Series Biological Sciences, vol.218, pp. 345-348, 1983.

[40]  F.E. Round, R.M. Crawford and D.G. Mann, The diatoms. Biology and morphology of the genera, Camgridge University Press, Camgridge, p. 747, 1990.

[41]  G.J.C. Underwood and D.M. Paterson, “Recovery of intertidal benthic diatoms after biocide treatment and associated sediment dynamics,” J. Mar. Biol. Assoc.U.K., vol. 73, pp. 25-45, 1993.

[42]  K. Wenderoth, J. Marquardt and E. Rhiel, “The big trail: many migrate at the expense of a few,” Diatom Res., vol. 19, pp.115–122, 2004.

[43]  J.A. Raven, “Small is beautiful, the picophytoplankton,” Funct. Ecol., vol. 12, pp. 503–513, 1998.


 



[1] The Ssame species resource as offor our tested diatoms, (i.e. the Nakdong River Estuary, isolated and supplied by the Korea Marine Microalgae Culture Center). and isolated from the Nakdong River estuary.


 [JH1]OR:

“microphytobenthic (MPB)”

 [JH2]“different” is implicit—OR (if you prefer—same meaning): “different light intensities and temperatures”

 [JH3]OR (both here and throughout the document): {If you want to refer to this device by its TYPE}: the

 [JH4]… just for consistency with the main text

 [JH5](?) I noticed that you do not use “sp”  (I assume “species”) with closterium, only with Nit.—is this correct? If not, be sure to add “sp.” to the instances of “closterium” or delete “sp.” (as implicit) from all instances of “Nit.”

 [JH6](ok here—elsewhere, and often, “sp.” is unnecessary)

 [JH7]Should this be “photons"-? If so, be sure to make the necessary change here and throughout the document.

 [JH8]OR:

“mechanisms are”

 

--*passim….

 [JH9](inserted period)

 [JH10]OR (if you want to retain the “controlling factor” phrase): “a key controlling factor for”

 [JH11]OR (alternative meaning): “the main”

 [JH12]OR: {Undo.}

 [JH13]*? (my assumption)

 [JH14]… *? (same here)

 [JH15](?) OR:

“peak tide”

 [JH16]implicit

 [JH17]redundant

 [JH18]… just for consistency

 [JH19]?

 [JH20]implicit

 [JH21]… just for consistency in the paper

 [JH22](?) I assume that “in situ” is redundant here—if not, write “their in situ

 [JH23]OR (if both in the same flask):

“together in a 2 L flask”

 [JH24]OR (if only 2): Both

 [JH25]?

 [JH26]?

 [JH27]OR (alternative meaning—here and throughout the paper): small-circle

 [JH28]Already identified

 [JH29]redundant

 [JH30]… because of the following “conditions”

 [JH31]*OR:

“was monitored to trace”

 [JH32]… just to mirror your original order

 [JH33]OR:

“a greater number of”

 [JH34]?

 [JH35]*OR (alternative meaning): “a higher motility (almost twice the F0) than did Nit.

 [JH36]OR: six

 [JH37]implicit

 [JH38]Change “arbitary” on both y-axes to “arbitrary.”

 [JH39]In the Figure, for consistency, include “sp.” For both or for neither.

 [JH40]OR (alternative meaning): significantly

 [JH41]ok? OR: MPB

 [JH42](??) I couldn’t really understand the intended meaning here, especially of the last sentence.

 [JH43]OR (alternative meaning): “different responses, including motility, to light and temperature”

 [JH44]OR (if you are still talking about “[33]” here): that

 [JH45](this is correct)

 [JH46]i.e. in the present study / OR (if you are speaking generally here): “have no effect on small species like Nit.

 [JH47]OR (alternative meaning):

“could help to further elucidate”

 [JH48]repetition

 [JH49]Neither included in word count nor checked

 [JH50](?) Check footnote again.

 [JH51]In the footnote, change the capital-E (in Estuary) back to lowercase if necessary.