MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY DOCTORAL DISSERTATION SUMMARY Major: Aquaculture Major code: 9 62 62 03 01 LE THI HONG GAM EFFECTS OF NITRITE, TEMPERATURE AND
Trang 1MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY
DOCTORAL DISSERTATION SUMMARY
Major: Aquaculture Major code: 9 62 62 03 01
LE THI HONG GAM
EFFECTS OF NITRITE, TEMPERATURE AND HYPERCAPNIA
ON PHYSIOLOGICAL PROCESSES AND GROWTH IN CLOWN KNIFEFISH
(Chitala ornata, Gray 1831)
Can Tho, 2018
Trang 2THE RESEARCH WAS PERFORMED AND COMPLETED AT CAN THO UNIVERSITY
Supervisor: Prof Dr Nguyen Thanh Phuong
Co-supervisor: Assoc Prof Dr Mark Bayley
The dissertation will be defended at the Doctoral Dissertation Assessment Committee at the University Level
At: ……….………
Time & Date:………
Reviewer 1: ………
Reviewer 2: ………
The dissertation can be found at:
The Learning Resource Center, Can Tho University
The National Library of Vietnam
Trang 3THE LIST OF PUBLISHED PAPERS
1 Gam, L.T.H., Jensen, F.B., Damsgaard, C., Huong,
D.T.T., Phuong, N.T and Bayley, M., 2017 Extreme
nitrite tolerance in the clown knifefish Chitala ornata is
linked to up-regulation of methaemoglobin reductase
activity Aquatic Toxicology 187: 9–17
2 Gam, L.T.H., Jensen, F.B., Huong, D.T.T., Phuong,
N.T., and Bayley, M., 2018 The effects of elevated environmental CO2 on nitrite uptake in the air-breathing
clown knifefish, Chitala ornata Aquatic Toxicology
196: 124-131
3 Gam, L.T.H., Vu, N.T.T., Nhu, P.N., Phuong, N.T and
Huong, D.T.T., 2018 Effects of nitrite exposure on haematological parameters and growth in clown knifefish
(Chitala ornata, Gray1831) Can Tho University Journal
of Science 54(2): 1-8
Trang 4CHAPTER 1 INTRODUCTION 1.1 Background
Climate change is defined as a change of climate that affected directly
or indirectly human activity, replacing the composition of the global atmosphere, and natural climate change recorded over long-term comparable periods of time (UNFCCC, 1992) This change has been caused by the increases of toxic gases such as CO2, N20, CH4 and green house gas concentrations as well as a temperature rise of 2.5 degrees Fahrenheit (1-4 degrees Celsius) over the next century (IPCC, 2013) According to the evaluation of vulnerability, Vietnam had the 27th rank among 132 countries over the world, which is under the impacts of climate change
With topographic characteristics and natural geographical conditions, the Mekong Delta (MD) becomes one of the areas having the most impacts over the world The increases of temperature induce the rise of metabolism of organism and aquatic animals as well as decomposition
of toxic compounds In the other hand, with the abundance of intensive culture system, overfeeding with waste products from excretion of aquatic animals has caused toxic gases such as: nitrite, carbon dioxide, ammonia, hydro sulfur…Especially, nitrite which is a product of nitrogen cycle, formed from ammonia in the condition of low dissolved oxygen level is well-documented toxin in aquatic system because it causes a lowering of blood oxygen with methaemoglobin formation (brown blood phenomenon), then leading a disturbance of respiration, physiological processes and growth (Kroupova et al., 2005) However, there have been a limited number of studies about effects of these environmental parameters to biological features, physiological processes in air-breathers, which may be seriously influenced by global climate change with their air-breathing activity To date only two
studies exist in air-breathers in the striped catfish (Pangasionodon
hypophthalmus) reported by Lefevre et al., 2011 and the snakehead
(Channa striata) also reported by Lefevre et al., 2012 with typical
results driven by high tolerance of nitrite in reducing nitrite uptake via gills and efficient denitrification mechanisms Besides, there have recently been several studies about effects of other environmental
factors in air-breathing fish such as Damsgaard et al (2015) about effects of carbon dioxide on acid-base regulation in P hypophthalmus
with high capacity of acid-base regulation compared to other breathing species Moreover, there is obviously not only one toxin existing in aquatic environment; the best assumption is that the
Trang 5air-combination of a variety of toxin may cause more bad effects by competition to uptake into fish blood
The facultative air-breathing C ornata is an important species in aquaculture throughout South East Asia C ornata is not only of high
commercial value as a source of protein for human consumption, but it
is also a costly ornamental fish species in tropical aquaria Therefore, the present dissertation about “Effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish
(Chitala ornata, Gray 1831)” was necessarily conducted to have an
understanding about effects and adaption mechanisms of this breathing fish under climate change
air-1.2 The objectives of dissertation
The objectives of this dissertation were to investigate the effects of nitrite, high concentrations of carbon dioxide and elevated temperatures
to physiological parameters and growth of the air-breathing C ornata
during sub-lethal and chronic exposures of these factors in isolation and combination in order to provide a better physiological understanding, particularly recommendations and solutions for minimizing impacts of nitrite toxicity and its combination with other environmental elements
in aquaculture ponds under global climate change
1.3 The main projects of dissertation
a) Conducting a survey on some selected environmental parameters
in C ornata ponds
b) Determining the 96 h LC50 of nitrite and examining the effect of
nitrite on haematological parameters and growth in C ornata
c) Determining the activity of metHb reductase in metHb reduction in
sub-lethal nitrite exposures in C ornata
d) Investigating the combined effect of nitrite and hypercapnia (high
concentration of carbon dioxide in the water) on haematological
parameters in small-sized and large sized C ornata
e) Determining the temperature tolerance and the effect of various
levels of temperature on haematological parameters in small-sized
and large sized C ornata
f) Determining 96 h LC50 of nitrite at elevated temperatures and investigating the effects of nitrite at different temperature on
haematological paramters in C ornata
g) Examining the effects of nitrite at different temperatures on haematological parameters, growth and digestive enzyme activity
in C ornata
1.4 The hypotheses of dissertation
Trang 6a) During nitrite exposure, C ornata reduce their branchial HCO3/Clexchanging rate and/or increase the activity of erythrocyte NADH metHb reductase for metHb reduction and experience significant changes in exchanging rate of branchial ions for recovery
b) pH regulation under a respiratory acidosis stimulate a reduction in branchial HCO3-/Cl- exchanger and thereby protect against nitrite
to blood cells, Hb and plasma ions in C ornata
e) C ornata has low tolerance of nitrite in the elevation of
temperature, leading to more significant effects to physiological parameters and growth compared to those in isolated exposure of
nitrite or isolated elevated temperatures
1.5 Significant contributions and applicability of the dissertation
The dissertation provides a better understanding about physiological
knowledge of the air-breathing clown knifefish C ornata including
recommendations and solutions for minimizing nitrite toxicity as well as its combination with other environmental elements in aquaculture ponds
under global climate change
With high tolerances of nitrite, temperature and hypercapnia in both
sub-lethal and chronic levels, C ornata can properly adapt with
extreme environmental changes such as temperature (24-33ºC), partial pressure of carbon dioxide (below 21 mmHg) and nitrite concentration (below 2.5 mM) contributing to the sustainable development of aquatic animals in the increases of temperature (1- 4ºC) in the next century and accumulation of toxic gases such as nitrite, carbon dioxide in intensive farming systems
The results of dissertation will be reliable background for conducting
deeper further studies about physiology in C ornata, other
air-breathing species or comparing with physiological responses of this species to those in other aquatic animals under extreme environmental changes
CHAPTER 2 METHODOLOGY
Trang 72.1 Project 1: Extreme nitrite tolerance in the clown knifefish
Chitala ornata is linked to up-regulation of methaemoglobin
reductase activity
Determination of acute nitrite toxicity (96 h LC 50 ): Fish (8-10g, n=576)
were randomly distributed to 48 tanks (36-L water each) and 12 fish per tank The fish were fasted for 2 days before exposure to 0, 2.6, 3.7, 4.8, 5.9; 7.0, 8.0, 9.1, 10.2, 11.3, 12.4 or 13.5 mM nitrite, with four replicate tanks for each concentration
Sub-lethal exposures: Fish (n=300, body mass 31.8 ± 1.8g) were
randomly taken from the 1m3 holding tank in a recirculation system with optimal water and placed in 200L experimental tanks with aerated water two days before experimentation Fish were fasted from this time until experiment termination Sub-lethal nitrite exposure concentrations were control, 1 mM and 2.5 mM, with one tank per treatment (100 fish per tank) Extra nitrite was added during the experiment to maintain the chosen concentration Ten fish were sampled from each tank at days 0,
1, 2, 3, 4, 5, 6 and 7
- Blood sampling: Blood was sampled by caudal puncture The fish
were placed on ice (which causes gentle initiation of a comatose state in this species) and 1mL of blood was withdrawn by from the caudal vein, using a heparinized syringe The fish were subsequently euthanized by severing the spine The blood was divided into two parts Half was used immediately for measurements of haemoglobin derivatives, haematocrit (Hct, ratio between volume of red blood cells), mean corpuscular haemoglobin concentration (MCHC, Huong and Tu, 2010) and extracellular pH (pHe), carbon dioxide tension (pCO2) and lactate, using the iSTAT analyzer (i-STAT Corporation, Princeton, USA) with CG4+ cartridges The remainder of the blood was centrifuged, and the plasma
was stored at -80°C for subsequent analysis of ions and osmolality The
values for pH and PCO2, HCO3- in the blood (Boutilier et al., 1985;
Cameron, 1971)
- Analysis procedures
+ Haemoglobin derivatives: The concentrations of oxyHb, deoxyHb,
metHb, HbNO were calculated by spectral deconvolution, following the
procedure described in Jensen (2007), Lefevre et al (2012) and Hvas et
al (2016) using reference spectra prepared from C ornata blood + Plasma ions: Plasma was obtained by centrifuging blood at 6000g for
6 min to determine the osmolality and the concentrations of Na+, Cl-,
NO2-, NO3- and protein Total osmolality was measured on a Fiske ten osmometer (Fiske® Associates, Two Technology Way, Norwood,
Trang 8one-Massachusetts, USA) Plasma concentration of Na was measured using
a flame photometer (Sherwood Model 420, Sherwood Scientific Ltd., Cambridge, UK) Plasma Cl- concentration was measured using a chloride titrator (Sherwood model 926S MK II Chloride analyzer) Plasma NO2- and NO3- was measured spectophotometrically using the
Griess reaction (Miranda, 2001; Jensen, 2007; Lefevre et al., 2011)
+ Plasma protein and measurement of whole body water content:
Plasma protein concentration was measured spectrophotometrically with Bio-rad protein assay (Bio-Rad Laboratory, Richmond, CA), using bovine serum albumin as standard (Bradford, 1976) Total body water was calculated from wet and dry weights of the fish The dry weight was determined by drying the fish at 60ºC until constant weight (for 4 days)
Methaemoglobin reductase activity: A series of fasted (2 days) fish
(n=216, body mass of 28-30 g) were exposed to 0, 1 and 2.5 mM nitrite, with each concentration replicated 6 times (6 tanks per treatment) The fish were sampled for blood at times 0, 2 and 6 days In control tanks, 6 fish were sampled at each sampling time, whereas three fish were sampled at each sampling in the nitrite tanks During the experiment, the water nitrite concentration was checked twice daily Blood (1.5 mL) was withdrawn from each fish of the exposure groups and washed four times (as above) with Ringer to obtain 3 mL nitrite-cleaned RBC suspension This RBC suspension was equilibrated to 1% CO2 with 99% air, and the MetHb decay was followed (as described above), where
after k in exposed fish was calculated
Statistics: All figures were made in Sigma plot 12.5 All data were
analyzed with PASW statistics (SPSS 18) Predicted mean, upper and lower 95% confidence intervals for the 96h LC50 were analyzed in JMP 9.0, using a logistic model A two- way ANOVA (the Holm-Sidak multiple comparison method, pair-wise comparison) was used to identify differences between treatments and sampling times for all parameters related to nitrite exposure (Hb derivatives, Hct, Hb, MCHC, plasma nitrite, plasma nitrate, plasma ions, plasma protein, osmolality and body water content) Normal distribution was tested using the Shapiro-Wilk test and where necessary data were log transformed to
achieve normality A p value of less than 5% (p<0.05) was judged
significant In the methemoglobin reductase activity experiments, the log of methemoglobin data was analyzed with linear regression to determine the slope for calculation of the rate constant All data are shown as standard error of the mean (S.E.M)
2.2 Project 2: Effects of nitrite exposure on haematological
parameters and growth in clown knifefish (Chitala ornata)
Trang 9Fish: (11.93±0.81 g; 11.53±0.15 g); Chemical: NaNO2 (Merck)
Effects of nitrite on haematological parameters: Fish (initial weight of
11.93±0.81 g, n=800) were randomly collected from 1 m3 holding tanks with optimal water quality, and subsequently distributed to 16 500-L tanks (200 L water contained and 50 fish per tank) The water in these tanks was continuously aerated in two days prior experimentation From the nitrite tolerance of clown knifefish (96 h LC50 of 7.82 mM nitrite,
Gam et al., 2017), the physiological experiment included 4 treatments
such as control, 0.2 mM (9.2 mg/L, recommended concentration), 0.4
mM (18.4 mg/L, 5% of 96 h LC50), and 4 mM nitrite (184 mg/L, 50% of
96 h LC50), with 4 replicates (4 tanks) for each treatment Nitrite in the water was recorded twice a day, and extra nitrite was added to maintain desirable concentrations during experimentation by spectrophotometer
using the Griess reaction (Lefervre et al., 2011; 2012) Three fish per
tank were sampled from each tank at days 0, 1, 3, 7, and 14 The ice was used for a comatose situation in fish before sampling blood A total volume of 300 µL of blood was collected from the caudal vein of each fish by a heparinised syringe for measuring haematological parameters
including: Hb and metHb (Jensen, 2007; Lefevre et al., 2011, 2012; Hvas et al., 2016; Gam et al., 2018)
Effects of nitrite on growth of C ornata:
- Method: Fish (initial weight 11.53±0.15 g, n=600) were randomly
taken from 1 m3 holding tanks with optimal water quality and subsequently distributed to 12 500-L tanks (300 L water and 50 fish per tank) with aerated water two days before experimentation The experiment included 4 treatments such as 0 mM (control), 0.2 mM, 0.4
mM, and 4 mM nitrite, with 3 replicates (3 tanks) for each treatment in
90 culturing days Nitrite in the water was recorded every three days before exchanging water (30%), and subsequently extra nitrite was added for maintaining the chosen concentrations The fish were fed with commercial pellets with feeding rate of 5% of body weight Humidity of commercial pellets (Shrimp feed with 38% protein, Tomboy Aquafeed Company, Vietnam) was less than 10% The pellets had uniform size (1
g = 203 pellets) Uneaten feed after 30 minutes of feeding was calculated for determination of feed used Thirty fish per tank were sampled on the days 0, 30, 60 and 90 for measuring growth parameters including WG, DWG, SGR, FCR, and SR
Statistics: Similar to Project 1 However, a one-way ANOVA was used
to identify differences between control treatment compared to other treatments for all growth parameters
Trang 102.3 Project 3: The effects of elevated environmental CO 2 on nitrite
uptake in the air-breathing clown knifefish Chitala ornata
Fish: C ornata (571±56.3 g); Chemicals: NaNO2, CO2 gas
Animal holdding and setup: C ornata from a local intensive farm were
transported to Can Tho University They were held at ambient laboratory temperature 27-28ºC in 4 cubic meter tanks with constant aeration (dissolved oxygen >90%) for 2 weeks before experimentation Fish were fed by commercial feed (shrimp feed with 38% protein, Tomboy Aquafeed company, Vietnam) Thirty percent of tank water was changed every second day to maintain optimal environmental condition (NO2- < 1 µM, NO3- < 40 µM and NH3 < 40 µM) Feeding was stopped 2 days before starting the experiment The experiment was performed in accordance with national guidelines on the protection and care of experimental in Vietnam A total of 24 fish were used
- Fish cannulation: They were anaesthetized in 0.05 g L-1 benzocaine and a polyethylene PE40 catheter (Smiths Medical International Ldt., Kent, UK) was inserted into the dorsal aorta through the dorsal side of
the mouth (Soivio et al., 1975), while the gills were irrigated with
well-oxygenated water containing 0.025 g L-1 benzocaine Fish recovered in well-aerated water for 24 h before starting experimentation to allow post-operative normalization of blood gasses
(Phuong et al., 2017a)
- Blood sampling: The experimental set-up included a large 500-L
tank from which water was re-circulated to 6 smaller 120-L tanks with 1 cannulated fish in each The water PCO2 was controlled with an Oxyguard Pacific system coupled with a G10ps CO2 probe and a K01svpld pH probe (Oxyguard International A/S, Farum, Denmark), which supplied CO2 to the water when pH changed above a value corresponding to the desired PCO2 in the water There were 4 exposure groups: (i) normocapnia (PCO2 < 0.7 mmHg); (ii) hypercapnia (PCO2 =
21 mmHg); (iii) 1 mM nitrite in normocapnic water and (iv) combined hypercapnia (acclimated hypercapnia) and 1mM nitrite In this combined group, the fish were cannulated then acclimated to hypercapnia (21 mmHg CO2) for 96 h before adding 1 mM nitrite Water temperature was controlled at 27-28ºC throughout experiments and water PO2 was above 120 mmHg Nitrite was added as NaNO2 and tested after each sampling time During the exposures, a volume of 0.8
mL blood was withdrawn from the catherter at 0, 3, 6, 24, 48, 72 and 96
h The blood was divided into two parts Half was used immediately for measurements of haematological parameters including: Hct, pHe, PCO2, and Hb derivatives The remainder of the blood was centrifuged (6 min
at 6,000g), and the plasma was stored at -80°C for subsequent analysis
Trang 11of plasma ions (NO2, NO3, HCO3, Na , K, Cl), glucose and osmolality
- Analytical procedures: all above haematological parameters were
measured with similar protocols described in Project 1
- Statistics: Similar methods to Project 1
2.4 Project 4: The combined effects of nitrite and elevated
clown knifefish (Chitala ornata)
Fish: C ornata juveniles (30-40 g); Chemicals: NaNO2, CO2 gas
- Animal holding and setup: Similar to Project 4, with small-size fish,
the density and blood sampling method were different Each treatment included 3 replicates (3 tanks, 45 fish/tank)
- Blood sampling: Blood samples (three fish per tank) were taken at 0,
3, 6, 24, 48, 72 and 96 h Fish were put in the ice for maintaining a comatose situation before sampling blood by a heparined (400 µL of blood per fish) for measuring similar haematological parameters as described in Project 3
- Analytical procedures: all above haematological parameters were
measured with similar protocols described in Project 1
- Statistics: Similar to Project 1
2.5 Project 5: Effects of different temperatures on haematological
parameters in clown knifefish (Chitala ornata)
Fish: (8-10 g, 29.2±3.4 g, 521 ± 32g); Materials: heater, cooler devices
Determination of temperature limits in C ornata: Twelve fish (8-10 g)
are prepared in plastic bucket (30 L water contained) The experiment consists of 2 groups: upper limit - increasing temperature and lower limit - decreasing temperatures with 6 replicates each The initial temperature was 27ºC; then it was increased or decreased (1ºC per hour)
by warm water, then using the heaters to maintain the desirable high temperature; or using iced water to decrease temperature, then using the coolers to maintain the desirable low temperature The fish was recorded swimming performance until 50% of the fish refusing respiration (no gill ventilation, no moving around) The upper and lower ranges of temperature were determined at that time
Effects of different temperatures on physiological parameters in sized C ornata: Fish (n=575, body mass of 29.2±3.4 g) were randomly
small-taken from the 1 m3 holding tank in a recirculation system with optimal water parameters and placed in 200-L experimental tanks with continued aeration Fish were fasted from this time until experiment termination The experiment included five levels of temperature: 24, 27,
Trang 1230, 33, and 36ºC with three tanks per treatment (45 fish per tank) After collecting the blood samples at day 0, desirable temperatures were adjusted by heaters or coolers (decreasing or increasing temperature 2ºC per 12 h) for blood sampling at day 1, 2, 3, 4, 5, 6, 7, and 14 (3 fish/tank) The fish were placed on ice, and then withdrawn by from the caudal vein (1 mL of blood for each fish), using a heparinized syringe The fish were subsequently euthanized by serving the spine for recognizing with other fish and maintaining the same stocking density
Effects of different temperatures on physiological parameters in sized C ornata: Fish (n=36, body mass of 521 ± 32g)
large Fish canulation: Similar to Project 3
- Experimental setup and blood sampling: The experiment was similarly
set up in 200L tanks with similar treatments to above experiment in
small-sized C ornata However, 1 cannulated fish in each, 6 tanks (6
replicates) per treatment, and a volume of 0.7 mL blood was withdrawn from the catheters at day 0, 1, 2, 3, 4, and 7
- Analytical procedures: In the blood: Hct, pH, PCO2; in the plasma: ions (NO2-, NO3-, HCO3-, Na+, K+, Cl-) and osmolailty These parameters were measured with similar methods described in Project 1
- Statistics: Similar to Project 1
2.6 Project 6: Effects of nitrite at different temperatures on
haematological parameters and growth in clown knifefish Chitala
ornata
Fish: (8-10g, 30-40g); Materials & chemicals: heaters, coolers, NaNO2
- Animal holding: Similar to Project 2
- Determination of acute nitrite toxicity (96 h LC 50 ) at 30ºC and 33ºC in
C ornata: Fish (8-10 g, n=1152) The two systems at 30ºC and 33ºC
were conducted by the same concentrations to determination of 96 h
LC50 for nitrite at 27ºC (Project 1, Gam et al., 2017) Heaters were used
for controlling temperatures in systems
- Sub-lethal nitrite exposures at different temperatures and blood sampling in C ornata:
- Experimental method: Fish (30-40 g, n=675) placed in 200- L
experimental tanks with aerated water two days before experimentation Fish were fasted from this time until experiment termination The experiment was conducted including 5 treatments: 1 mM nitrite at 24,
27, 30, 33, and 36ºC with three tanks per treatment (45 fish per tank) After collecting the blood samples at day 0, chosen temperatures were adjusted by heaters (decreasing or increasing temperature 2ºC per 12 h) Nitrite was subsequently added, and blood samples collected at day 1; 2;
Trang 133; 4; 5; 6; 7; and 14 (3 fish/tank) After placing the fish on ice (which causes gentle initiation of a comatose state in this species), 1 mL of blood was withdrawn by from the caudal vein, using a heparinized syringe The fish were subsequently euthanized by serving the spine for recognizing with other fish and maintaining the same stocking density
- Analytical procedures: All haematological parameters chosen and their
analyzing methods were similar as described in Project 1
- Effects of nitrite at different temperatures on growth and digestive enzyme activities in C ornata:
- Experimental method: Fish (8-10 g, n=900) were placed in 500-L
experimental tanks with aerated water two days before experimentation The experiment was randomly conducted including 6 treatments: 27ºC; 30ºC; 33ºC; 1 mM nitrite at 27ºC; 1 mM nitrite at 30ºC; and 1 mM nitrite at 33ºC with 3 tanks/treatment (50 fish per tank) After collecting growth samples at day 0, the temperatures were adjusted by heaters until reaching the chosen temperatures (2ºC per 12 h) Nitrite was subsequently added, and growth samples collected at day 30, 60, and 90 for measuring growth parameters such as DWG, SGR, SR, and FCR (thirty fish randomly sampled in each tank) Three fish per tank were collected intestine and stomach at day 90 for examining activities of digestive enzymes During experimentation, the fish were fed twice a day (the mixture of trashfish and commercial feed (Shrimp feed with 38% protein, Tomboy Aquafeed company) used in the first month, and then only commercial feed used until the end of growth experiment After 30 minutes feeding, uneaten feed was removed for counting the total number of pellets and weighed trashed fish used, then subtracting their humidity to obtain the actual weight of feed used Dead fish was removed every day, and thirty percent of the tank water was changed every third day to maintain optimal environmental conditions Nitrite concentration was checked before exchanging water for supplementing the lacked amount of nitrite
- Analytical procedures: growth parameters were measured by similar
methods as described in Project 1; digestive enzymes such as pepsine
(Worthington, 1982), trypsine (Tseng et al., 1982), chymotrypsine
(Worthington, 1982), α-Amylase (Bernfeld, 1951)
- Statistics: Similar to Project 1 However, a one-way ANOVA was
used to identify differences between control treatment compared to other treatments for all growth parameters and digestive enzyme
2.7 Project 7: A survey on some environmental parameters in clown
knifefish (Chitala ornata, Gray 1831) ponds