Publisher’s note: this comment is a copy of AC3 and its content was therefore removed.

• This manuscript investigated stable isotopic compositions and protein expression patterns of the vent crabs inhabiting the yellow vents and the white vents in famous shallow-water hydrothermal vents located off Kueishan Island, Taiwan. This manuscript provided little isotope and protein expression data (n=16) compared to previous literature. It does not match the general data quality/quantity for Biogeosciences. Due to the limited data, the calculated isotopic niche width for the vent crabs from different vent types may not be representative. The discrimination of protein expressions may also be problematic.

Reply:

Thanks so much for all of your valuable comments. We actually conducted the isotopic and proteomic studies twice in 2010. We collected vent crabs on July 2 at both vents, Aug. 4 at WV, and 24 at YV. The specimens used in the isotope niche width and proteomic analyses differed in samples of July but were the same in August. Our primary aim was to compare the feeding habits of different vent types. So, we decided to report the results from August samples only to avoid any potential influence by inter-individual variations. Although the data were not too many, we think the results are still significant.

Here, we combined the two sets of data to investigate spatial and temporal variations in the feeding habits of the vent crabs. We found that crabs' δ¹³C and δ¹⁵N values significantly differed spatially and temporally (MANOVA test, p < 0.05). The niche width of vent crabs from YV-Aug (0.88 ‰²) narrowed substantially compared to other groups (i.e., YV-July (2.94 ‰²), WV-July (2.88 ‰²), and WV-Aug (3.62 ‰²)) (p<0.05), respectively. The protein expression patterns of the crabs exhibited three groups, i.e., WV-July & YV-July, WV-Aug, and YV-Aug, respectively. Our results indicated that the dwelling crabs were associated with their living vent, and within-vent variability was more noticeable in YV compared to WV. We revised the MS, and the main reanalyzed results are as follows.

Table 2. The isotopic data and statistical results of vent crabs (Xenograpsus testudinatus) from the white and yellow vents in July and August 2010. (a) The δ¹³C and δ¹⁵N values of vent crabs; (b) results of the two-way multivariate analysis of variance (MANOVA, Pillai's trace). W: white vent; Y: yellow vent; black bold: p<0.05; sampling date: July 2 (0702), August 4 (0804), and August 24 (0824); n: sample size. 

(a)

(b)

Table 3. The ellipses analyses of vent crabs (Xenograpsus testudinatus) from the white and yellow vents in July and August 2010. (a) Comparisons of the SEAc areas among crab groups using Layman metrics based on the posterior distribution (95% credited intervals) of the modes (p<0.05, A>B); (b) the overlapping percentage of ellipses area among groups. W: white vent; Y: yellow vent; SEAc: standard ellipse area corrected; sampling date: July 2 (0702), August 4 (0804), and August 24 (0824).

(a)

(b)

Figure 6. Results from the combined principal component analysis (PCA) and cluster analysis of Bray–Curtis similarity (BCS) indices using standardized overall protein expressions. W: white vent; Y: yellow vent; m: male; f: female; band 1–27: variable of protein bands; 0702: July 2; 0804: August 4; 0824: August 24.

• The discussion should have discussed the data more thoughtfully. I can only get more details of related data suggesting the consistency among various studies (in 4.1, 4.2). Or there are more summaries of similar observations demonstrating this work is not unique (in 4.2, 4.3). It’s also difficult to follow the logic (in 4.4). Such criticism may result from the author’s writing skills, preventing my understanding.

Reply:

Thanks so much for your comments. We also extensively revised our discussion. The corrected sections (4.2, 4.3, and 4.4) are as follows.

4.2 The isotopic niche width of vent crabs from the WV and YV

Wu et al. (2021a) and Hung et al. (2019) reported that the δ¹³C and δ¹⁵N values of vent crabs significantly differed between WV and YV. However, both studies combined specimens from two sampling months. Wu et al. conducted their experiments in July and August 2010, with the values of -17.4 ± 0.2 ‰ (WV; n = 44) and -16.3 ± 0.2 ‰ (YV; n = 17) for δ¹³C and 7.8 ± 0.14 ‰ (WV) vs. 6.7 ± 0.3 ‰ (YV) for δ¹⁵N, respectively (Wu et al., 2021a). Hung et al. gathered their samples in April and July 2010. They found male crabs from YV differed from all other groups, i.e., YV-female, WV-male, and WV-female, respectively (sample size and data not shown) (Hung et al., 2019).

Within-vent variability in δ¹³C and δ¹⁵N values of vent crabs was also documented in several studies. Hung et al. collected their samples in April and July 2010, and the δ¹³C and δ¹⁵N values of both male and female crabs exhibited no difference between the center and edge of a WV (sample size and data not shown) (Hung et al., 2019). In Wang et al., crabs from one site influenced by both WV and YV and three peripheral groups (150–300 m) presented a wide range of δ¹³C (-20.5 to -14.3 ‰) and δ¹⁵N (3.2 to 9.8 ‰) values sampled in June and July 2014 (Wang et al., 2022). And, there was no significant difference in the isotopic data among the four groups (p > 0.05), i.e., -16.9 ± 0.77 ‰ and 8.1 ± 0.94 ‰ (n = 6); -17.2 ± 1.34 ‰ and 7.5 ± 1.01 ‰ (n = 40); -16.6 ± 1.03 ‰ and 7.2 ± 1.43 ‰ (n = 156); -16.9 ± 0.66 ‰ and 8.3 ± 1.17 ‰ (n = 10), respectively. Further isotopic niche analysis demonstrated that the contribution of dead zooplankton as a food source to those crabs ranged from > 34 % (vent center) to ≤ 18 % (peripheral sites). We also analyzed the isotopic data published by Chang et al. for comparison (Chang et al., 2018). They gathered vent crabs from a WV along the southwest transact in August and September 2015. The δ¹³C and δ¹⁵N values were significantly different between the center and periphery (70 – 100 m) (MANOVA, p = 0.01), i.e., -16.20 ± 2.49 ‰ and 5.33 ± 4.06 ‰ (n = 4); -17.55 ± 0.74 ‰ and 8.85 ± 0.79 ‰ (n = 10), respectively. Dead zooplankton as a food source for those crabs were 6 – 38 % vs. 16 – 42%, respectively.

In this study, the δ¹³C and δ¹⁵N values of vent crabs significantly differed between vent types and sampling months (MANOVA test, Table 2). Our results showed that the crabs’ isotopic niche width (shown as the SEAc area) was considerably narrower in YV-Aug (0.88 ‰²) than those in YV-July, WV-July, and WV-Aug (2.94, 2.88, and 3.62 ‰²) (p < 0.05), respectively (Table 3). In the southwest Mediterranean, seasonal variations in the δ¹³C and δ¹⁵N values of the sally lightfoot crab Percnon gibbesi ranged from -18.33 to -13.08 ‰ and from 3.71 to 8.2 ‰ in 2016 (Bada et al., 2022). The isotopic niche width of P. gibbesi varied from 1.4 ‰² in winter to 4.5 ‰² in autumn, while the data were 1.5 and 2 ‰² in spring and summer, respectively. It showed that the diets of P. gibbesi in autumn had the widest niche (food variability) linked to the local variability in algal resources. In the Pechora Sea, the isotopic niche width in scavenger hermit crab Pagurus pubescens varied between sites of 4N and 9N with a distance of 13 km because of a significant difference in their macrobenthic abundance (Gebruk et al., 2021). The isotopic niche width for the hermit crab was 0.15 ‰² at 4N and 0.27 ‰² at 9N, with 0.05 ‰² overlapped. Differences in diet sources were correlated with local macrobenthic clams as shown at 4N, characterized by low Astarte montagui (32 g m-2), in contrast to the high biomass of A. borealis and Macoma calcarea (500 g m^-2) at 9N. The niche width of this hermit crab had an even smaller overlapping SEAc area than our between-vent comparisons, i.e., 1.47 ‰² in July and 0.86 ‰² in August. In brief, our study clearly shows that the isotopic signatures of the resident vent crabs reflected temporal and spatial heterogeneities. The discrepant results among different studies indicate explicit state sampling information, including size, date, and location, is essential.

4.3 Protein expression patterns of vent crabs from the WV and YV

Our proteomic results indicated that vent crabs were distinguishable as groups of WV-July & YV-July, WV-Aug, and YV-Aug, respectively. In the case of dove snails, A. misera inhabiting in WVs of KS Islet, their protein expression patterns were related to the diffusion of locally emitted vent fluids (Chen et al., 2015). The naturally acidified seawater in the southward sampling site had pH ranges from 7.78 to 7.82, while it was 7.31–7.83 in the east, southwest, and northwest locations. Based on the expressed protein profiles, the Anachis snails were classified into the south and another group. In a CO₂-SV off Vulcano Island in Sicily, sea anemones Anemonia viridis were collected at a distance of 350 – 800 m from a vent, where the pH values were 7.6, 7.9, and 8.2, respectively (Urbarova et al., 2019). Gene expression patterns of A. viridis revealed two clades, i.e., low pH group (pH 7.6) vs. high pH ones (pH 7.9 and pH 8.2). Overall, mobile vent crabs, slow-moving dove snails, and sessile sea anemones all performed adaptation abilities associated with their environments.

Organisms respond to environmental changes in a time-dependent manner. When the Chinese mitten crabs E. sinensis were transferred to high salinity (25 psu) for six days, the protein profiles of posterior gills were different from the control group (0 psu) (Yang et al., 2022). The nutrition value of linoleic acid (18:2n-6, LA) and α-linolenic acid (18:3n-3, LNA) in the Chinese mitten crabs E. sinensis was evaluated in the laboratory for 107 days (Wei et al., 2018). A total of 186 proteins were expressed differentially in the hepatopancreas between the groups of LA and LNA. In the Teboulba fishing harbor in Tunisia, high levels of aliphatic and aromatic hydrocarbon pollutants were in the sediments (Jebali et al., 2014). The Mediterranean crabs C. maenas showed differential protein expression patterns in hepatopancreas between control (day 0) and exposed groups with 15, 30, and 60 days. These proteomic-based studies exhibited the earliest responses of tested crabs to environmental changes detected at least on day 6. In this study, the protein expression patterns of vent crabs changed in one month (Fig. 5), indicating the vent environments probably fluctuated often.

4.4 Association of crabs’ feeding habits with vent types

It has long been known that WVs and YVs in KS Islet differ in the color and composition of vent plumes (Chen et al., 2005b; Lebrato et al., 2019; Mei et al., 2022). A relatively low fluid temperature and high pH in WVs compared to YVs (30–65 vs. 54 - 121 °C and 1.84–6.96 vs. 1.52-6.32 (pH seawater scale, 25 °C) (Table 1). Recently, Lebrato et al. studied temporal biogeochemical changes in this SV system during 2009 – 2018 (Lebrato et al., 2019). Their principal findings are the catastrophic earthquake and typhoon Nepartak in 2016 shaped the seabed morphology, seawater chemistry, vent fluid composition and flow rate, and benthic ecology, then gradually recovered in 2018. In addition, the reduction in venting activity and fluid flow in YV was more severe than in WV. Similarly, the temperature fluctuation ranges of our sampling WV were relatively small compared to those in YV, i.e., 47 – 62 vs. 54 - 121 °C, respectively (Table 1).

Previous studies reported that the movement of vent crabs reveals different spatial scales. The daily foraging movement is in the vent area (Jeng et al., 2004; Chang et al., 2018; Allen et al., 2020). During the reproductive season, ovigerous females move to the vent periphery, release their larvae, and then return to the chimneys (Hung et al., 2019). The migratory distance was about 100–200 m horizontally from the vent mouth. Besides, vent crabs were absent in the by-catch of nearby non-vent fisheries (Wang et al., 2013). And the holotype of this species was collected from a 15 m deep rocky reef in the Gengxin Fish Port, Peikuan, I-Lan County, Taiwan (Ng et al., 2000). These investigations indicate that vent crabs can actively move and survive in vent and non-vent environments. However, how far and how often the crabs move around is unknown. Here, we demonstrated the vent crabs exhibited temporal and spatial variations in isotopic niche width and protein expression patterns (Table 3 and Fig. 6). Even with a distance of 100 m; the endemic vent crabs are strongly associated with their vent types. In addition, within-vent variability in food sources is more dramatic in YV compared to WV.

Additional references:

Bada, N., Da Ros, Z., Rindi, F., Busi, S., Azzurro, E., Derbal, F., Fanelli, E.: Seasonal trophic ecology of the invasive crab Percnon gibbesi (Brachyura, Plagusiidae) in the southwestern Mediterranean: Insights from stomach contents and stable isotope analyses. Mar. Environ. Res., 173, 105513, doi:10.1016/j.marenvres.2021.105513, 2022.

Lebrato, M., Wang, Y. V., Tseng, L. C., Achterberg, E. P., Chen, X. G., Molinero, J. C., Bremer, K., Westernstroer, U., Soding, E., Dahms, H. U., Kuter, M., Heinath, V., Johnck, J., Konstantinou, K. I., Yang, Y. J., Hwang, J. S., Garbe-Schonberg, D.: Earthquake and typhoon trigger unprecedented transient shifts in shallow hydrothermal vents biogeochemistry, Sci. Rep., 9(1), 16926, doi: 10.1038/s41598-019-53314-y, 2019.

Wu, J. Y., Lin, S. Y., Peng, S. H., Hung, J. J., Chen, C. T. A., Liu, L. L.: Data on isotopic niche differentiation in benthic consumers from shallow-water hydrothermal vents and nearby non-vent rocky reefs in northeastern Taiwan. Data Br., 37, 107216. doi:10.1016/j.dib.2021.107216, 2021b.

• Several specific comments are as followings.

1. Table 1 needs to be clarified and easier to read. The geochemical data for vent fluids from previous literature need to be synthesized more appropriately.

   Reply:

   Thanks so much for your comments. The revised Table 1 is as follows.

   Table 1. Location and environmental measurements of the study sites. (Mean ± S.E.); n: sample size.

   Environmental parameters		WV (White vent)	YV (Yellow vent)	Sampling date	References
   Shallow-water hydrothermal vents		WVs	YVs
   Vent plume
   	Temperature (°C)	30-65 (50.7 ± 8.2, n = 109); 31-38	78-116 (106 ± 9.16, n = 115); 50-90	2000; 2017	Chen et al., 2005b; Mei et al., 2022
   	pH	1.84-6.96  (3.2 ± 1.17, n = 110)	1.52-6.32 (2.49 ± 0.72, n = 116)	2000	Chen et al., 2005b
   	H2S  (mmol mol-1)	2.3-21.0 (12.94 ± 4.55, n = 4)	7.6-114.7 (60.12 ± 19.57, n = 6)	“	“
   	CO2  (mmol mol-1)	916-987 (n = 3)	976-992 (n = 2)	“	“
   	N2  (mmol mol-1)	0.02-0.04 (n = 3)	0.11-2.23 (n = 2)	“	“
   Sampling vent’s geographic coordinates		24.83404° N, 121.96172° E	24.83553° N, 121.96361° E
   Vent plume
   	Temperature  (°C)	47-49 (48.00 ±0 .37, n = 6); 55 ± 4	115-116 (115.40 ± 0.22, n = 5); 106 ± 6	2010-2014; 2010-2011	Chen et al., 2016; Hung et al., 2019
   		62	97	2010.07.02	Lin, 2011
   		41	105	2010.08.03-05	Yang et al., 2012
   		58	97	2010.08.24-27	“
   			65; 105; 121; 105; 54-63	2009; 2010.08.07; 2011; 2016.03; 2016.08-2017.08	Lebrato et al., 2019
   	pH	5.45 ± 0.65	2.48 ± 1.06	2010-2011	Hung et al., 2019; Lin, 2011
   		5.06	2.81	2010.07.02	Lin, 2011
   		4.83	2.82	2010.08.03-05	Yang et al., 2012
   		5.74	2.22	2010.08.24-27	“
   	H2S  (mmol mol-1)	2.2-57.4 (18.4 ± 8.4, n = 6)	4.3-172.4 (90.8 ± 29.1, n = 6)	2010-2014	Chen et al., 2016
   	CO2  (mmol mol-1)	161.7-760.6 (503.8 ± 78.7, n = 8)	731-881.6 (798.4 ± 23.8, n = 6)	“	“
   	N2  (mmol mol-1)	109.5-633.7 (309.9 ± 72.4, n = 8)	33.4-140.9 (65.1 ± 17.0, n = 6)	“	“
   Crab collecting site
   	Distance to vent center (m)	~ 5	~ 5	2010.08	This study (WV: 0804; YV:0824)
   	Depth (m)	17	7	“	“
   	Temperature (°C)	25	26.7	“	“
   	pH	7.3	7.8	“	“
   	Deposited sulfur particles (diameter)	globules (~ 0.05–0.1 cm)	balls (> 2 cm)	“	“

1. The drawing quality of figures 3 and 4 need to be improved.

   Reply:

   Thanks so much for your comments. Figure 3 is the output of Excel in Microsoft, and Figure 4 is the output of the analysis software SIBER v2.1.6 (Stable Isotope Bayesian Ellipses in R) package in R 4.2.2 software (R Development Core Team, 2013) and RStudio 2022.12.0-353. The following ones are the best quality we got.

   

Figure 3. The δ¹³C and δ¹⁵N values of vent crabs (Xenograpsus testudinatus) from the white and yellow vents. W: white vent; Y: yellow vent; sampling date: July 2 (0702), August 4 (0804), and August 24 (0824); m: male; f: female; the crabs with label: same individuals for proteomic experiments.

Figure 4. Convex hull and standard ellipses areas based on the δ¹³C and δ¹⁵N values of vent crabs (Xenograpsus testudinatus) from the white and yellow vents. Dot lines: convex hull areas; solid lines: standard ellipses areas (SEAc); W: white vent; Y: yellow vent; 0702: July 2; 0804: August 4; 0824: August 24.

1. Geochemical and isotopic data are mentioned repeatedly.

Reply:

Thanks so much for your comments. We analyzed more data and did our best to avoid repetitive discussion in our revised MS.

1. The estimate of isotopic niche width for vent crabs need to include plenty of isotope data collected in previous studies (e.g. Wu et al., 2021).

Reply:

Thanks so much for your comments. We have already included additional data in our revised MS, and the main findings are presented above.

Publisher’s note: this comment is a copy of AC4 and its content was therefore removed.

General comments:

This study investigated stable isotope niche width, and protein expression for vent crab Xenograpsus testudinatus from the shallow water hydrothermal vents located off Kueishan Islet, Taiwan. To do this, authors provided total of 16 samples, nine from the white vent (WV) and seven from the yellow vent (YV) for comparison. In addition, authors also compared the benthic community between the two habitats using the quadrates along four transects. However, in my opinion, the scientific quality and rigors of this manuscript do not fulfil the requirement of Biogeosciences for the following reasons:

1. There is no clear hypothesis or research question. Specifically, the authors failed to demonstrate why it is important to investigate the endemic vent crabs in different vent types in this shallow-water hydrothermal vent environment. It is unclear why this would be interesting for the scientific community to gain this knowledge. In addition, since protein expression depends on a variety of factors such as physiological (e.g. stress) and environmental condition (e.g. pollution, pH, etc.), I would assume the protein results would be different because the YV and WV conditions are vastly different, so where do the authors go with this information? 

Reply:

I apologize for not seeing your valuable comments in the supplementary file. We actually conducted the isotopic and proteomic studies twice in 2010. Related explanations and major revised results are presented in reply to Referee #1 and our revised MS. Vent crabs can move to a distance of 100 – 200 m, as shown in ovigerous females. However, their regular moving range is unknown. If trans-vent movement is common, we expect to have no significant difference between WV and YV by the application of proteomic tools. We also revised our abstract to point out the significance of our study. Here is the revised abstract.

The shallow-water hydrothermal vents (SVs) located off Kueishan (KS) Islet, Taiwan, are one of the world's most intensively studied vent systems. It has long been known that white vents (WVs) and yellow vents (YVs) differ in the color and composition of vent plumes. The endemic vent crabs (Xenograpsus testudinatus) are abundant in both vent types, and ovigerous females migrate to the vent periphery with a distance of 100 – 200 m to release their offspring. However, most research on the vent crabs was associated with WV or unspecified vent areas. To increase our knowledge of crabs dwelling in other vent types, we compared the feeding habits of vent crabs living in WV and YV with two sampling months. Specifically, we examined the benthic community of WV and YV, isotopic niche width, and protein expression patterns of the crabs from the two vents at a distance of 100 m and sampled in July and August 2010. The coverage of sessile organisms and low-mobility fauna in WV was more abundant than those in YV based on the survey in August 2010. The δ¹³C and δ¹⁵N values of crabs significantly differed spatially and temporally (MANOVA test, p < 0.05). The niche width of vent crabs from YV-Aug (0.88 ‰²) narrowed substantially compared to the rest, i.e., YV-July (2.94 ‰²), WV-July (2.88 ‰²), and WV-Aug (3.62 ‰²) (p<0.05), respectively. Based on the protein expression patterns, the vent crabs exhibited three groups, i.e., WV-July & YV-July, WV-Aug, and YV-Aug, respectively. Our results indicated that the dwelling crabs were associated with their living vent, and within-vent variability was more noticeable in YV compared to WV. We suggested that vent crabs inhabit their resident vent. Even at a scale of meters, trans-vent movement is probably rare as an adaptation to minimize predation risk.

2. Sample sizes are too small for robust inference. Given that the main question in the paper is to examine whether the isotope niche widths of X. testudinatus from two vent types are similar, total of 16 samples are too small for this. Although authors used the corrected standard ellipse area (SEAc) to lessen the biases towards smaller sample sizes, I suspect additional data will change the SEAc and overlapped SEAc as well. The authors should discuss any potential biases due to the sample sizes and how the niche width may change when sample sizes are increased in the discussion.

Reply:

Thanks so much for your comments. We added more data to investigate spatial and temporal variations in the feeding habits of the vent crabs. Our data indicate sample size and inter-individual variation are both important factors. For comparative purposes, we present the isotopic and proteomic results with the same data sets grouped by vent types vs. vent types and sampling months.

• Isotopic results based on vent types.

• Proteomic results based on vent types.

• Isotopic results based on vent types and sampling months.

• Proteomic results based on vent types and sampling months.

1. Data needs more thoughtful interpretation. Several paragraphs in the discussion were written like results. The authors listed some previous literature without properly connected to the interpretation of their own data. For example, section 4.2 paragraph 1: The isotope values between the YV and the WV from this study were not significant different, but a previous study by Wu et al., (2021) did show differences in isotope values between the YV and WV in different sampling year. The authors mentioned Wu et al.’s work, but never discussed what factors might have contributed the different outcomes between the two studies. The authors also mentioned another study that compared isotope values of vent crabs between different sex but also did not connect to their own study. Another example, in section 4.2 paragraph 3, the authors compared the isotope niche overlap percentages observed in the vent crabs with hermit crabs from a totally different environment (in Pechora Sea). However, it is difficult to see why the hermit crab is relevant to the study site in KS. This part should be better explained or deleted.

Reply:

Thanks so much for your comments. We extensively revised our discussion, and the corrected sections (4.2, 4.3, and 4.4)  as follows.

4.2 The isotopic niche width of vent crabs from the WV and YV

Wu et al. (2021a) and Hung et al. (2019) reported that the δ¹³C and δ¹⁵N values of vent crabs significantly differed between WV and YV. However, both studies combined specimens from two sampling months. Wu et al. conducted their experiments in July and August 2010, with the values of -17.4 ± 0.2 ‰ (WV; n = 44) and -16.3 ± 0.2 ‰ (YV; n = 17) for δ¹³C and 7.8 ± 0.14 ‰ (WV) vs. 6.7 ± 0.3 ‰ (YV) for δ¹⁵N, respectively (Wu et al., 2021a). Hung et al. gathered their samples in April and July 2010. They found male crabs from YV differed from all other groups, i.e., YV-female, WV-male, and WV-female, respectively (sample size and data not shown) (Hung et al., 2019).

Within-vent variability in δ¹³C and δ¹⁵N values of vent crabs was also documented in several studies. Hung et al. collected their samples in April and July 2010, and the δ¹³C and δ¹⁵N values of both male and female crabs exhibited no difference between the center and edge of a WV (sample size and data not shown) (Hung et al., 2019). In Wang et al., crabs from one site influenced by both WV and YV and three peripheral groups (150–300 m) presented a wide range of δ¹³C (-20.5 to -14.3 ‰) and δ¹⁵N (3.2 to 9.8 ‰) values sampled in June and July 2014 (Wang et al., 2022). And, there was no significant difference in the isotopic data among the four groups (p > 0.05), i.e., -16.9 ± 0.77 ‰ and 8.1 ± 0.94 ‰ (n = 6); -17.2 ± 1.34 ‰ and 7.5 ± 1.01 ‰ (n = 40); -16.6 ± 1.03 ‰ and 7.2 ± 1.43 ‰ (n = 156); -16.9 ± 0.66 ‰ and 8.3 ± 1.17 ‰ (n = 10), respectively. Further isotopic niche analysis demonstrated that the contribution of dead zooplankton as a food source to those crabs ranged from > 34 % (vent center) to ≤ 18 % (peripheral sites). We also analyzed the isotopic data published by Chang et al. for comparison (Chang et al., 2018). They gathered vent crabs from a WV along the southwest transact in August and September 2015. The δ¹³C and δ¹⁵N values were significantly different between the center and periphery (70 – 100 m) (MANOVA, p = 0.01), i.e., -16.20 ± 2.49 ‰ and 5.33 ± 4.06 ‰ (n = 4); -17.55 ± 0.74 ‰ and 8.85 ± 0.79 ‰ (n = 10), respectively. Dead zooplankton as a food source for those crabs were 6 – 38 % vs. 16 – 42%, respectively.

In this study, the δ¹³C and δ¹⁵N values of vent crabs significantly differed between vent types and sampling months (MANOVA test, Table 2). Our results showed that the crabs’ isotopic niche width (shown as the SEAc area) was considerably narrower in YV-Aug (0.88 ‰²) than those in YV-July, WV-July, and WV-Aug (2.94, 2.88, and 3.62 ‰²) (p < 0.05), respectively (Table 3). In the southwest Mediterranean, seasonal variations in the δ¹³C and δ¹⁵N values of the sally lightfoot crab Percnon gibbesi ranged from -18.33 to -13.08 ‰ and from 3.71 to 8.2 ‰ in 2016 (Bada et al., 2022). The isotopic niche width of P. gibbesi varied from 1.4 ‰² in winter to 4.5 ‰² in autumn, while the data were 1.5 and 2 ‰² in spring and summer, respectively. It showed that the diets of P. gibbesi in autumn had the widest niche (food variability) linked to the local variability in algal resources. In the Pechora Sea, the isotopic niche width in scavenger hermit crab Pagurus pubescens varied between sites of 4N and 9N with a distance of 13 km because of a significant difference in their macrobenthic abundance (Gebruk et al., 2021). The isotopic niche width for the hermit crab was 0.15 ‰² at 4N and 0.27 ‰² at 9N, with 0.05 ‰² overlapped. Differences in diet sources were correlated with local macrobenthic clams as shown at 4N, characterized by low Astarte montagui (32 g m-2), in contrast to the high biomass of A. borealis and Macoma calcarea (500 g m^-2) at 9N. The niche width of this hermit crab had an even smaller overlapping SEAc area than our between-vent comparisons, i.e., 1.47 ‰² in July and 0.86 ‰² in August. In brief, our study clearly shows that the isotopic signatures of the resident vent crabs reflected temporal and spatial heterogeneities. The discrepant results among different studies indicate explicit state sampling information, including size, date, and location, is essential.

4.3 Protein expression patterns of vent crabs from the WV and YV

Our proteomic results indicated that vent crabs were distinguishable as groups of WV-July & YV-July, WV-Aug, and YV-Aug, respectively. In the case of dove snails, A. misera inhabiting in WVs of KS Islet, their protein expression patterns were related to the diffusion of locally emitted vent fluids (Chen et al., 2015). The naturally acidified seawater in the southward sampling site had pH ranges from 7.78 to 7.82, while it was 7.31–7.83 in the east, southwest, and northwest locations. Based on the expressed protein profiles, the Anachis snails were classified into the south and another group. In a CO₂-SV off Vulcano Island in Sicily, sea anemones Anemonia viridis were collected at a distance of 350 – 800 m from a vent, where the pH values were 7.6, 7.9, and 8.2, respectively (Urbarova et al., 2019). Gene expression patterns of A. viridis revealed two clades, i.e., low pH group (pH 7.6) vs. high pH ones (pH 7.9 and pH 8.2). Overall, mobile vent crabs, slow-moving dove snails, and sessile sea anemones all performed adaptation abilities associated with their environments.

Organisms respond to environmental changes in a time-dependent manner. When the Chinese mitten crabs E. sinensis were transferred to high salinity (25 psu) for six days, the protein profiles of posterior gills were different from the control group (0 psu) (Yang et al., 2022). The nutrition value of linoleic acid (18:2n-6, LA) and α-linolenic acid (18:3n-3, LNA) in the Chinese mitten crabs E. sinensis was evaluated in the laboratory for 107 days (Wei et al., 2018). A total of 186 proteins were expressed differentially in the hepatopancreas between the groups of LA and LNA. In the Teboulba fishing harbor in Tunisia, high levels of aliphatic and aromatic hydrocarbon pollutants were in the sediments (Jebali et al., 2014). The Mediterranean crabs C. maenas showed differential protein expression patterns in hepatopancreas between control (day 0) and exposed groups with 15, 30, and 60 days. These proteomic-based studies exhibited the earliest responses of tested crabs to environmental changes detected at least on day 6. In this study, the protein expression patterns of vent crabs changed in one month (Fig. 5), indicating the vent environments probably fluctuated often.

4.4 Association of crabs’ feeding habits with vent types

It has long been known that WVs and YVs in KS Islet differ in the color and composition of vent plumes (Chen et al., 2005b; Lebrato et al., 2019; Mei et al., 2022). A relatively low fluid temperature and high pH in WVs compared to YVs (30–65 vs. 54 - 121 °C and 1.84–6.96 vs. 1.52-6.32 (pH seawater scale, 25 °C) (Table 1). Recently, Lebrato et al. studied temporal biogeochemical changes in this SV system during 2009 – 2018 (Lebrato et al., 2019). Their principal findings are the catastrophic earthquake and typhoon Nepartak in 2016 shaped the seabed morphology, seawater chemistry, vent fluid composition and flow rate, and benthic ecology, then gradually recovered in 2018. In addition, the reduction in venting activity and fluid flow in YV was more severe than in WV. Similarly, the temperature fluctuation ranges of our sampling WV were relatively small compared to those in YV, i.e., 47 – 62 vs. 54 - 121 °C, respectively (Table 1).

Previous studies reported that the movement of vent crabs reveals different spatial scales. The daily foraging movement is in the vent area (Jeng et al., 2004; Chang et al., 2018; Allen et al., 2020). During the reproductive season, ovigerous females move to the vent periphery, release their larvae, and then return to the chimneys (Hung et al., 2019). The migratory distance was about 100–200 m horizontally from the vent mouth. Besides, vent crabs were absent in the by-catch of nearby non-vent fisheries (Wang et al., 2013). And the holotype of this species was collected from a 15 m deep rocky reef in the Gengxin Fish Port, Peikuan, I-Lan County, Taiwan (Ng et al., 2000). These investigations indicate that vent crabs can actively move and survive in vent and non-vent environments. However, how far and how often the crabs move around is unknown. Here, we demonstrated the vent crabs exhibited temporal and spatial variations in isotopic niche width and protein expression patterns (Table 3 and Fig. 6). Even with a distance of 100 m; the endemic vent crabs are strongly associated with their vent types. In addition, within-vent variability in food sources is more dramatic in YV compared to WV. 

1. The writing needs an overhaul: A) The Introduction is unfocused and did not present a clear hypothesis. The stable isotope and proteome methods in the Introduction seem to be out of place. B) There are a lot of repetitive geochemical information for the KS vent region in the Introduction and Method. Some of these values (e.g. temperature ranges, etc) don’t even match. Similarly, there are a lot of repetitive information in the Results and Interpretation for the isotope niche width. This information needs to be condensed and streamlined. C) There are numerous unclear sentences in the manuscript, e.g. line 102-104, 105-107, 200-204 etc. D) The authors cited some previous work, but required readers to go into the original references and figure out what they mean, e.g. line 207-209: 4N and 9N.

Reply:

Thanks so much for your comments. We revised the whole MS. Major results (L200-204 & L207-209 in 4.2 The isotopic niche width of vent crabs from the WV and YV) and discussion are presented above. Some of the revised introduction parts are as follows.

Stable isotope analysis is commonly applied in the study of animal feeding ecology. Through the processes of assimilation, consumers increase with stable isotope values of 0.0–1.3 ‰ for δ¹³C and 1.4–5 ‰ for δ¹⁵N in each trophic transfer (DeNiro and Epstein, 1978, 1981; Post, 2002; McCutchan et al., 2003). With the isotopic data, consumers' trophic position and niche width can be calculated (Layman et al., 2011). Trophic studies in SVs in KS Islet revealed that dead zooplankton killed by sulfur plumes (as plankton-derived production) is essential to scavengers and carnivores based on the δ¹³C and δ¹⁵N data (Wang et al., 2014; Chang et al., 2018; Wu et al., 2021a). The importance of dead zooplankton to vent crabs decreases from the vent center to the periphery (Wang et al., 2022). Furthermore, vent crabs collected from YV had significantly lower δ¹³C and δ¹⁵N values than those in WV (Wu et al., 2021a).

The proteome is the set of expressed proteins in an organism, which varies with tissue, physiological condition, and environment where the organism lives. By proteomic tools, the difference in protein expression profiles of the studied organisms under stresses or changing environments can be characterized (Lopez-Pedrouso et al., 2020). For example, the variation of protein patterns of the dove snail A. misera was consistent with the diffusion of local vent fluids in KS Islet (Chen et al., 2015). Proteomic studies exhibited differential expression signatures in the Chinese mitten crab (Eriocheir sinensis) when treated with different feeds (Wei et al., 2018) or hyper-osmotic stress (Yang et al., 2022), in mud crab Scylla olivacea when exposed to heavy metals (Razali et al., 2019), and in Mediterranean crab (Carcinus maenas) from different harbors (Jebali et al., 2014). Similarly, we can extend our knowledge of the within- and between vents’ variations of protein patterns of the crabs living in SVs by applying proteomic tools.

Although the vent crab (X. testudinatus) is one of the most intensively studied species in SV systems, most research was associated with WV or unspecified vent areas. Studies on crabs dwelling in other vent types are rare. Therefore, spatial and temporal variations in the feeding habits of vent crabs were investigated in this study. Specifically, we examined the benthic community of WV and YV, isotopic niche width, and protein expression patterns of the crabs from two vents at a distance of 100 m and sampled in July and August 2010.

Specific comments:

Line 51: Change “more depleted” to “lower”. You can say “something is depleted in 13C” but you can not say “depleted d13C values”. The correct way is to say “lower d13C values”

Reply:

Thanks so much for your comments. We corrected the usage according to your suggestion.

Line 59: Reference Lopez-Pedrouso et al., is missing in reference list.

Reply:

Thanks so much for your comments. We included the reference in our revised MS.

Added reference:

López-Pedrouso, M., Varela, Z., Franco, D., Fernández, J. A., Aboal, J. R.: Can proteomics contribute to biomonitoring of aquatic pollution? A critical review, Environ. Pollut., 267, 115473, doi:10.1016/j.envpol.2020.115473, 2020.

Line 96: Unclear, please rephrase.

Reply:

Thanks so much for your comments. The revised paragraph is as follows.

2.3 Preparation of vent crabs for isotope niche width and proteomic studies

Vent crabs have gathered 5 m away from the mouths of the WV and YV on sampling dates of July 2 (both vents), August 4 (WV), and 24 (YV) 2010, respectively. The specimens used in the isotope niche width and proteomic studies differed in samples of July but were the same in August. Each collected crab was covered with aluminum foil and kept in liquid nitrogen, then frozen at -80 °C for later use. Crab samples were examined for cleaning debris, and epibionts, then their carapace width and wet weight were measured before dissection (Fan et al., 2016).

Line 116: The standard ellipse area is SEA, the corrected standard ellipse area is SEAc. Please change.

Reply:

Thanks so much for your comments. The corrected sentence is as follows.

Measurements of isotopic niche width, proposed by Layman et al. (2007), were calculated for vent crabs, e.g., the corrected standard ellipse area (SEAc), which was a measure of the mean score of the isotopic niche occupied by all crab individuals in each group and their potential primary food sources in the δ¹³C and δ¹⁵N space (Jackson et al., 2011).

Line 151: Change “insignificantly different” to “not significantly different”. This expression has been used in several places, e.g. line 18-19, line 187, please change them all.

Reply:

Thanks so much for your comments. We corrected the usage according to your suggestion.

Line 164, Remove “the first study”. This is not the first study to investigate the feeding habits of vent crab (X. testudinatus) in the KS vent sites (see Wang et al., 2020 https://www.biorxiv.org/content/10.1101/2020.09.09.288985v1.full). Also, this kind of claim should be avoided in general.

Reply:

Thanks so much for your comments. The corrected sentence is as follows.

This study compared the feeding habits of vent crabs (X. testudinatus) from a WV and a YV within 100 m.

Line 196: Change “insignificant differences” to “no significant differences”

Thanks so much for your comments. We corrected the usage according to your suggestion.

Line 200: Changed to “varied from 18-34%”

Reply:

Thanks so much for your comments. The corrected sentence is as follows.

Further isotopic niche analysis demonstrated that the contribution of dead zooplankton as a food source to those crabs ranged from > 34 % (vent center) to ≤ 18 % (peripheral sites).

Line 205: I am unclear why this reference in Pechora Sea is relevant to the study. The environment is completely different.

Line 206: The 4N and 9 N are study sites from the cited reference, but readers have to find the original paper to get this information. Again, it is unclear how the study site in Pechora Sea is relevant to KS site.

Reply:

Thanks so much for your comments. We revised the paragraph in 4.2 The isotopic niche width of vent crabs from the WV and YV, which is presented above.

Line 212-220, See my general comments about interpretation.

Reply:

Thanks so much for your comments. We revised the paragraphs in 4.3 Protein expression patterns of vent crabs from the WV and YV, which is presented above.

Line 243: Remove “the first study”. This kind of claim should be avoided in general.

Reply:

Thanks so much for your comments. The corrected sentence is as follows.

This study compared the benthic community, isotopic niche width, and protein expression patterns of the endemic vent crabs (Xenograpsus testudinatus) from different types of SVs at 100 m.