Genotype-environment interactions for survival and growth rate at varying levels of sodium chloride for growth hormone transgenic channel catfish (Ictalurus punctatus), channel catfish, and albino channel catfish

Journal Pre-proof Genotype-environment interactions for survival and growth rate at varying levels of sodium chloride for growth hormone transgenic channel catfish (Ictalurus punctatus), channel catfish, and albino channel catfish

Nermeen Y. Abass, David Drescher, Nathan Backenstose, Zhi Ye, Khoi Vo, Ramjie Odin, Guyu Qin, Sheng Dong, Rex A. Dunham PII:

S0044-8486(18)31681-8

DOI:

https://doi.org/10.1016/j.aquaculture.2020.735084

Reference:

AQUA 735084

To appear in:

aquaculture

Received date:

3 August 2018

Revised date:

28 January 2020

Accepted date:

4 February 2020

Please cite this article as: N.Y. Abass, D. Drescher, N. Backenstose, et al., Genotypeenvironment interactions for survival and growth rate at varying levels of sodium chloride for growth hormone transgenic channel catfish (Ictalurus punctatus), channel catfish, and albino channel catfish, aquaculture (2019), https://doi.org/10.1016/ j.aquaculture.2020.735084

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© 2019 Published by Elsevier.

Journal Pre-proof Genotype-environment interactions for survival and growth rate at varying levels of sodium chloride for growth hormone transgenic channel catfish (Ictalurus punctatus), channel catfish, and albino channel catfish Nermeen Y. Abass

a,b

Vo a, Ramjie Odin a

a,c

, David Drescher

School of Fisheries,

a,f

, Nathan Backen stose

, Guyu Qin a, Sheng Dong

Aquaculture and

a,d

a,e

, Zhi Ye

, Khoi

a,g

, Rex A. Dunham a*

Aquatic Sciences,

Auburn University, AL

36849, USA of

Agricultural

Botany,

Faculty

of

Agriculture

Saba-Basha,

f

Department

oo

b

c

current

address:

Department

of

Animal

address:

Department

of

NY 14260- 1300, USA current

address: Department

address:

Mindanao

State

Jo u

current

Sciences,

of Biochemistry,

rn

WA 98195, USA f

Biological

al

e

Avian

Sciences,

SUNY

University

of

Buffalo,

Buffalo,

University of Washington,

Seattle,

Maguindanao,

Sinsuat,

Pr

current

and

e-

Maryland, Colleg e Park, MD 20742, USA d

pr

Alexa ndria University, Alexandria City, P.O. Box 2153, Egypt

University,

Datu

Odin

Magu indanao 9601, Philippines g

current

address:

Department

of

Biosystems

Engineering,

Auburn

University,

Aubu rn, AL 36849, USA *Corresponding author: Tel.: + 1 334 844 9121; fax: + 1 334 844 9208. E-mail address: [email protected] (R.A. Dunham)

Abstract Lack of freshwater is emerging as the most critical natural resource issue facing

humanity.

Ongoing

climate

change 1

will

reduce

freshwater

supplies,

and

Journal Pre-proof demand

for food

punctatus, driven

fry

by

promoter

transgenic

the

(rtMT-ccGH),

continues to expand rapidly. Swim-up channel catfish, Ictalurus

or

for

rainbow by

trout

the

(opAFP-ccGH),

the

channel

catfish

Oncorhynchus

ocean

pout

channel catfish,

growth

mykiss

Zoarces and

hormone

metallothionein

americanus

albino

(GH)

gene

promoter

antifreeze

protein

channel catfish were grown

at 0, 2.5, 5, and 7.5 parts per thousand (ppt) salinity. Survival was 100% for all genetic groups at 0 and 2.5 ppt. Increasing salinity to 5 ppt decreased overall rtMT-ccGH control (C),

opAFP-ccGH

transgenic

(T),

opAFP-ccGH

oo

f

survival as survival rates of rtMT-ccGH transgenic (T), control

(C),

channel

catfish,

and

pr

albino channel catfish were 83, 82, 83, 77, 89 and 40%, respectively. Increasing

e-

salinity to 7.5 ppt had a strong negative impact on survival as means for rtMT-

Pr

ccGH (T), rtMT-ccGH (C), opAFP-ccGH (T), opAFP-ccGH (C), channel catfish, and albino channel catfish were 67, 13, 67, 10, 18, and 7%, respectively with the channel catfish

having

the

lowest

al

albino

survival followed

by opAFP-ccGH (C)

rn

(P = 0.002). Raising salinity to 2.5 ppt greatly increased the growth rate of GH channel catfish

(11-33

Jo u

transgenic

%),

channel catfish (56%),

and

albino

channel

catfish (124%). NaCl had a negative effect on survival and growth rate for swimup

fry at 5 ppt. Significant differences were observed at varying salinity (P <

0.0001) and

for

survival

occurred

final body

weights

were

perfectly

not

among

genetic

correlated,

for the overall phenotype.

groups.

and

Apparently,

Condition

factor,

genotype-environment

growth

interactions

GH can play a critical positive

role for channel catfish under osmotic stress, which has relevance for aquaculture management

under

future

genotypes was observed catfish were less affected

global

climate

change.

with slightly elevated,

2.5

Optimum ppt.

performance

for

all

Although, GH transgenic

than non-transgenic channel catfish at 5-7.5 ppt, their 2

Journal Pre-proof performance was sub-optimal. GH transgenic fish are beneficial for use in culture environments that have slight shifts in salinity in the future, but they are only a partial solution for futu re environments at 5 ppt and higher.

Keywords:

Transgenic channel catfish – Growth rate – Survival rate – Growth

Jo u

rn

al

Pr

e-

pr

oo

f

hormo ne (GH) – opAFP- ccGH – rtMT- ccGH.

3

Journal Pre-proof Intro duction Water

is the most critical issue for the survival of all living organisms.

Only 3% of the world’s water is freshwater (WWF, 2018). Increasing pressures on

freshwater

present

supplies

unprecedented

from agriculture, challenges

aquaculture,

(UNFPA,

industry,

2016).

and

Global

human

climate

activity

change

is

altering patterns of water around the world, causing shortage of freshwater and an increase in brackish water (EPA, 2015, 2016). The amount of water available in

oo

f

many areas is already limited, and demand for freshwater will continue to increase as population grows (EPA, 2016). By 2025, two-thirds of the world’s population face

shortage

of

freshwater

(WWF,

and the

global

population

is

e-

expected to reach 9.6 billion (FAO , 2016a).

2018),

pr

may

Pr

Aquaculture is one of the fastest growing animal food producing industries in the world (FAO, 2016b). Fish is a major source of animal protein and nutrition 2016b).

al

for people worldwide (FAO,

Catfish production accounts for the largest

rn

aquaculture output in the United States. Catfish production peaked at 300 million

and

2016,

University,

Jo u

kg in 2003 then declined to 226, 127, 138 and 150 million kg in 2007, 2008, 2011 respectively 2017).

The

(Hanson

majority

and of

Sites,

catfish

2014,

2015;

production

occurs

Mississippi in

the

State

states

of

Mississippi, Alabama, Arkansas, and Texas (USDA, 2018). The deficiency of freshwater resource in numerous countries leads to a shift to develop freshwater fishes in brackish water and seawater (El-Sayed, 2006; Yan et al., 2013). Obviously, most freshwater fish are not adapted to grow in these environments.

Growth hormone has a role in osmoregulation in fish (Almeida et

al., 2013).

4

Journal Pre-proof Growth hormone (GH) has been considered as a candidate gene for growth and development in fish (Tian et al., 2014), and increased expression of this gene could

be

advantageous

for

growing

fish

in

increasingly

brackish

environments.

Growth hormone (GH) is a pluripotent hormone produced by the pituitary gland in

teleosts,

and

acts

by

binding

to

a

single transmembrane receptor,

the GH

receptor (GHR) (Björnsson et al., 2002; Reinecke et al., 2005). Growth hormone (GH) gene transgenesis has been applied in several fish species to enhance growth

al.,

2008;

accelerated

Leggatt

growth

et

rates

al.

2012).

(Devlin

et

GH al.,

transgenesis 2000,

has

2015),

pr

et

oo

f

(Devlin et al., 1994, 1995, 1999; Morales et al., 2001; Dunham et al., 2002; Nam resulted

better

feed

in

greatly

conversion

e-

(Tibbetts et al., 2013), tolerance of low temperature (Abass et al., 2016), and

Sangiao-Alvarellos

et

al.,

et al., 1993; Varsamos et al., 2005;

Pr

increased resistance to salinity (Sakamoto

2005; Sakamoto

and

McCormick,

2006; Hallerman

et

al

al., 2007; Almeida et al., 2013).

rn

The objective of this study was to compare the growth and survival rate growth

hormone

(GH)

siblings,

control channel catfish,

Jo u

among

transgenic and

albino

channel

catfish,

channel catfish

non-transgenic at

varying

full

levels of

sodiu m chloride.

Mate rials and Methods Experimental fish Channel catfish (opAFP-ccGH antifreeze

and

protein

Ictalurus

rtMT-ccGH) promoter

punctatus growth hormone (GH) gene constructs driven

(opAFP)

metallothionein promoter (rtMT) (Fig.

by

the

ocean

or

rainbow

pout Zoarces americanus

trout

Oncorhynchus

mykiss

1) have been transferred to channel catfish 5

Journal Pre-proof I. punctatus via electroporation (Qin et al., 2016) to produce P1 transgenic GH catfish

the

transgene

generations. injection

All the

of

was

integrated,

experimental fish

luteinizing

expressed, were

hormone-releasing

and

produced

hormone

inherited

by

analog

in

induced

subsequent

spawning with

(LHRHa)

(Su

et

al.,

2013). One F1 (P1 transgenic male mated with a control female) family of channel catfish transgenic for the catfish growth hormone gene driven by the ocean pout Zoarces americanus antifreeze protein promoter (opAFP-ccGH),

one F1 (P1

f

with a control female) family of channel catfish transgenic

oo

transgenic male mated

and

for the catfish growth hormone gene driven by the rainbow trout Oncorhynchus metallothionein

promoter

(rtMT-ccGH).

One

pr

mykiss

one family of albino

produced

spawning.

hormone-induced

The

of

Kansas

Random

channel catfish were also

transgenic

opAFP-ccGH

and

Pr

by

e-

(KR) strain channel catfish and

family

rtMT- ccGH, and albin o channel catfish were from an unknown strain.

al

Fish handling and pre-challenge conditions

rn

After spawning, the fertilized egg masses were incubated in wire mesh

Jo u

baskets in paddle wheel troughs. Dead eggs were removed daily. The pH ranged from 7.0 to 7.3 and DO from 6.9 to 7.7 mg/L. Water flow through each tank was maintained at 15 L/min to ensure a renewal rate of at least twice per hour. Swim-up fry were first fed Artemia species (San Francisco Bay Brand, Inc. Newark, CA) and fry starter diet. A total of 180 swim-up fry from each genetic group (7dph) were randomly divided into groups of 45 fry per treatment. There was no mortality of fry prior to the initiation of the experiment. There were four salinity treatments (0 ppt, 2.5 ppt, 5 ppt, and 7.5 ppt). 15 swim-up fry of each genetic group were stocked in triplicate per 6

Journal Pre-proof treatment in circular 3-L plastic tanks. All fry were initially held at 0 ppt then sodium chloride was added to increase the salinity by 2.5 ppt / 3 days until the final treatment level was reached. The source of water was from ponds and was 0 ppt.

Everyday 70–80% of the total water was replaced with the

respective concentration of saline water. The pH ranged from 7.0 to 7.3, DO from 6.9 to 7.7 mg/L, temperature from 22 to 27 °C, and ammonia nitrogen

and

rtMT-ccGH

was

blind,

as

the

oo

opAFP-ccGH

f

kept at 0 ppm. Mortality was monitored daily for 30 days. The test for the transgenic

and non-

pr

transgenic full-siblings were mixed and not identified by PCR until the conclusion of the experiment. At the conclusion of the experiment, it was

e-

determined that 11-18% of the fry were transgenic in the transgenic group,

Pr

likely due to mosaicism in the parent P1 (Dunham, 2011). Commencing the day after they were introduced into the different salinity treatments, swim-up

al

fry were fed Aquamax fry powder twice a day to satiation (Cat#: 000-7684,

rn

Purina Mills, St. Louis, MO). Mean body weight and total length of swim-up

Jo u

fry were recorded at the end of the experiment. Dead fish were identified, counted and recorded on a daily basis. Survival of fry and condition factors (CF) were calculated as follows: Survival (%) =

(number of fry survived) × 100 initial number of fry

Condition factors (CF) =

100 × W L3

Where W is weight (g) and L is total length (cm). Screen ing of transgenic Genomic DNA extracted as described in the protocol of Kurita et al. (2004) with

some

modification.

The

quality

and 7

quantity

of

DNA

samples

were

Journal Pre-proof confirmed

using

DNA

Spectrophotometers. specific

primers

agarose

Transgenic

as described

gels

fish

and

samples

were

NanoDrop screened

2000/2000c

with

in the protocol of Abass et al.

PCR

(2016).

with

Primers

sequences were as follows: A (5′ to 3′) GCG ACT CTG TTC TGC ACA CG, B (5′ to 3′) ACC ACG CTC AGA TAG GTC TC, C (5′ to 3′) GCC AAG ATG ATG GAC GAC TT, D (5′ to 3′) AGG AAG CTC TGT TGC CTG AA, E (5′ to 3′) CCT CGC TCA AGG TCT GGT AG, F (5′ to 3′) TGA CCC GAC CTC AGA TAA GC, and G (5′ to 3′) CAA AGG TCT TAA GCG CAT CC (Fig. 1). products

were

visualized

on

oo

f

PCR

an

ethidium bromide

1.2%

TAE

agarose gel and

(Bio-Rad

Laboratories,

Inc).

The Amplified

e-

software

pr

documented with a Molecular Imager® Gel Doc™ XR+ System using Image Lab™ PCR products were purified

Pr

and sequenc ed as described in the protocol of Abass et al. (2016). Statis tical analysis

effects

MANOVA

rn

combined

within-subjects

of salinity,

time,

(repeated

and

measures)

genotype.

to

Due to

evaluate

the

a strong interaction

Jo u

multiway

al

Data on survival rate were expressed as mean ± SD, and subjected to

among the factors (salinity and time), the effect of each factor was tested at a fixed

level of the other factor using one-way ANOVA. Final body weight and

condition factors (K) were expressed as mean ± SD, and subjected to one-way ANOVA

at

fixed

genetics

groups

(Duncan,

1995)

levels

were at

P

of

salinity.

determined <

0.05.

using

Significant Duncan's

Statistical analyses

softw are (SAS Institute, 2010).

Results 8

differences multiple were

among

different

comparison

conducted

using

test SAS

Journal Pre-proof Influence of salinity on survival rate Salinity, time, and genetic group all affected survival (P < 0.0001). Additionally, time × genetic group (P = 0.01), salinity × time, and salinity × time × genetic group interactions occurred (P < 0.0001) (Table 1; Fig. 2). No significant mortality was recorded for any genetic groups at 0 and 2.5 ppt. However, large differences were observed at 7.5 ppt (Table 1; Fig. 2).

f

Increasing salinity to 5 ppt decreased survival rate, as survival rates of rtMT-

oo

ccGH transgenic (T), rtMT-ccGH control (C), opAFP-ccGH transgenic (T),

pr

opAFP-ccGH control (C), channel catfish, and albino channel catfish were 83.3, 82.4, 83.3, 76.9, 88.9, and 40%, respectively, (Table 1; Fig. 2) with the

e-

albino channel catfish having the lowest survival (40 %) (P = 0.046). Survival

Pr

was not different among transgenic groups and their controls, and channel catfish. Raising salinity to 7.5 ppt had a strong negative impact on survival

al

and means for rtMT-ccGH (T), rtMT-ccGH (C), opAFP-ccGH (T), opAFP-

rn

ccGH (C), channel catfish, and albino channel catfish were 66.7, 12.8, 66.7,

Jo u

10.1, 17.8, and 6.7%, respectively (Table 1; Fig. 2). A massive fish mortality occurred during the second week for most genotypes at 7.5 ppt. Mortality continued

for all genotypes, except that GH transgenics still had high

survival (66.7%)

(P =

0.002). Survival

of GH

transgenics

channel

catfish stabilized after the end of the second week (P = 0.01) (Table 1). Repeated measure MANOVA revealed that the factors salinity and time had significant effects on survival rates of the respective genetic groups, with significant interaction among these factors (P < 0.0001). Influence of salinity on growth rate

9

Journal Pre-proof Growth was also affected by salinity for ictlaurid catfish (P < 0.0001) (Supplementary Table 1; Fig. 3). Differences in final body weight were observed among each transgenic group and their control, channel catfish and albino channel catfish at varying salinity. No significant differences in final body weights were observed between rtMT-ccGH (T) and opAFP-ccGH (T) at 0, 5, and 7.5 ppt. However, there was a significant difference between

f

rtMT-ccGH (T) and opAFP-ccGH (T) at 2.5 ppt (Supplementary Table 1; Fig.

oo

3). Raising salinity to 2.5 ppt greatly increased the growth rate for rtMT-

pr

ccGH (T), rtMT-ccGH (C), opAFP-ccGH (T), opAFP-ccGH (C), channel catfish, and albino channel catfish by 33.3, 22.6, 10.9, 13.3, 55.6, and

e-

123.8%, respectively. However, increasing salinity to 7.5 ppt had a strong

Pr

negative impact and decreased the growth rate for rtMT-ccGH (T), rtMTccGH (C), opAFP-ccGH (T), opAFP-ccGH (C), channel catfish, and albino

al

channel catfish by 85, 83.9, 85. 5, 80, 77.8, and 76.2%, respectively

rn

(Supplementary Table 1; Figs 3 and 4). During the early part of experiment,

Jo u

the fish were gaining weight at 7.5 ppt and then reached point where they stopped growing and began to lose weight. However, the transgenic fish were much less effected than the controls. No significant difference in final body lengths were observed between rtMT-ccGH (T), and opAFP-ccGH (T) at 2.5, 5, and 7.5 ppt. However, there was a significant difference between rtMT-ccGH (T) and opAFP-ccGH (T) at 0 ppt (Supplementary Table 1).

Significant difference in final body length

were observed among each transgenic group and their controls, channel catfish

and

albino

channel

catfish

at

0

and

2.5

ppt

(P

<

0.0001)

(Supplementary Table 1). Increasing salinity to 7.5 ppt greatly decreased the 10

Journal Pre-proof body length for rtMT-ccGH (T), rtMT-ccGH (C), opAFP-ccGH (T), opAFPccGH (C), channel catfish, and albino channel catfish by 42.6, 40.2, 43.5, 48, 52.9, and 54.4%, respectively (Supplementary Table 1). Condition factor (CF) of GH transgenic channel catfish was similar to non- transgenic full siblings and all other control channel catfish, and albino channel catfish at 0 ppt and 7.5 ppt (P = 0.01), although albino channel

f

catfish had significantly higher CF than GH transgenic channel catfish, non-

oo

transgenic full siblings, and control channel catfish at 5 ppt (P < 0.0001) (Fig.

pr

5). CF of channel catfish had significantly higher than opAFP-ccGH(T), opAFP-ccGH(C), and control albino channel catfish at 2.5 ppt (P < 0.0001)

e-

(Fig. 5). Rate of body size gain peaked at 2.5 ppt and decreased at higher

Pr

salinity for all genetic groups (Fig. 4).

al

Discuss ion

freshwater

water

will

increase

Jo u

brackish

rn

The planet’s supply of freshwater is fixed, and the proportion of

will

intensify

due

as

to

climate

population and

change.

Competition

demand increases

for from

agriculture, industry and other urban activities. The combined effect of these factors will lead to shortages of freshwater. Genetically engineered fish that have high performance and that can be used in brackish water might help alleviate this problem. An alternative view is that this information is critical to assess the environmental risk of GH fish and catfish both in the present and in the future in conjunction with climate change. In

the

current

study,

we

transferred

channel

catfish Ictalurus

punctatus growth hormone cDNA construct driven by the ocean pout Zoarces 11

Journal Pre-proof americanus antifreeze trout Oncorhynchus

protein

promoter

(opAFP-ccGH)

mykiss metallothionein

promoter

or

rainbow

(rtMT-ccGH)

to

channel catfish to produce transgenic GH catfish and compared the survival and growth rate among transgenic GH catfish, non-transgenic controls, channel catfish, and albino channel catfish. As gene insertion commonly has pleiotropic effects, it is important to measure non-target traits of potential

f

economic and environmental impact.

As

the

salinity

increased

to 5 ppt, genotype × environment

pr

salinity.

oo

All genetic groups of catfish exhibited 100% survival at 0–2.5 ppt

interactions were apparent. Survival dramatically decreased for all genotypes,

their

potential

usefulness

for

future

Pr

to

e-

except rtMT-ccGH (T) and opAFP-ccGH (T) still had high survival, alluding climate

change.

Massive

mortality occurred for different genetic groups during the second week at 7.5

al

ppt except for GH transgenic channel catfish. The transgene was the main

rn

effect rather than the size of the fish. In one case, the GH fish were only 33%

Jo u

larger, but had much greater survival than the control. In the 7.5 ppt treatment the fish were very small, but survived. Some fish among both transgenic and non-transgenic fish were among the largest, yet experienced mortality. Allen and Avault (1971) found that fingerlings of channel catfish from two different locations (9.9 to 21.5 cm total length, 5.2 to 55.4 g in weight) at 14 ppt showed signs of distress early in the experiment, but showed some signs of recovery near the middle or end of experiment. This illustrates the importance of the length of these experiments. Additionally, salinity tolerance within a species appears to be complex and influenced by genetic type, size

12

Journal Pre-proof and geographic location, likely due to differences in the concentration of other ions at different locations. All freshwater and saltwater fishes exhibit Na+/K+-ATPase enzyme activity in the gill epithelia to maintain ion permeability in the cytoplasmic membrane, relative stability of various ion concentrations in the intracellular environment, and osmotic pressure balance between the intracellular and

is

(McCormick,

2001).

Generally,

ATPase

enzyme

f

activity

environments

positively correlated with the external salinity concentration

pr

(Tipsmark et al., 2002; Geng et al., 2016).

oo

external

The higher mortality of swim-up fry for all the genetic groups in 5 ppt

e-

and 7.5 ppt was probably due to osmoregulatory failure (Enayati et al., 2013;

Pr

Abass et al., 2017). In the early stages of development, fishes may not yet have effective mechanisms to eliminate excess Na+ and Cl− from their body (Ghahremanzadeh

et

al.,

al

systems

2014;

Abass

et

al.,

2017).

NaCl

rn

concentrations of 7.5 ppt appeared to elicit a toxic effect on the swim-up fry

Jo u

stages regardless of the exposure duration. At 7.5 ppt, GH transgenic channel catfish had 67% survival, which was higher than that of their transgenic full siblings and all other control channel catfish, and albino channel catfish. GH plays a role in osmoregulation (Almeida et al., 2013; Abass et al., 2016); thus, salinity tolerance of rtMTccGH (T) and opAFP-ccGH (T) might be expected to be altered. Growth hormone (GH) increased the capacity of brown trout, Salmo trutta, to tolerate exposure to seawater (Smith, 1956; Madsen, 1990) due to the capacity of this hormone to increase the number and size of gill chloride cells, Na+, K+ATPase,

and

the

Na+,

K+,2Cl-

cotransporter 13

(NKCC),

ion

transporters

Journal Pre-proof involved in salt secretion (McCormick, 2001; Pelis and McCormick, 2001; Sakamoto and McCormick, 2006). Plasma GH levels have been increased in stenohaline catfish following exposure to 12 ppt seawater (Drennon et al., 2003). Growth hormone regulates seawater-type chloride cells and associated biochemistry (Sakamoto and McCormick, 2006). It is not surprising that salinity tolerance of albinos was altered.

f

Pleiotropic have been observed for other traits in albino Silurus glanis catfish

catfish

can

exhibit reduced reproductive capacity, especially in

pr

Albino

oo

such as reduced aggressiveness and shoaling behavior (Slavík et al., 2016).

production affects osmoregulation.

e-

females (Dunham, 2011). It is not apparent how disruption of melanin

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The results of the present study showed that the growth rate for swimup fry in 2.5 ppt for all the genetic groups was better than at 0 ppt, 5 ppt, and

al

7.5 ppt. NaCl above 2.5 ppt for swim-up fry can cause some stress and

rn

decrease survival, appetite, growth rate, and even weight loss (Abass et al.,

Jo u

2017). At 5 ppt, growth and survival decreased, however, weight gain was less affected for rtMT-ccGH (T) and opAFP-ccGH (T) than rtMT-ccGH (C), opAFP-ccGH (C), channel catfish, and albino channel catfish. Genotypeenvironment interactions were prevalent. Genotype-environment interactions also were observed when fry of channel catfish, blue catfish, Ictalurus furcatus, and their hybrid, channel catfish female × blue catfish male, were grown at 0, 3 and 6 ppt NaCl (Abass et al., 2017). In that case, channel catfish and blue catfish growth rate was much better at 3 ppt than 0 ppt, and genotype-environment interactions were even greater than the current study as growth of channel catfish and blue catfish 14

Journal Pre-proof doubled at 3 ppt while hybrid catfish growth was increased slightly (Abass et al., 2017). At 6 ppt, heavy mortality occurred and growth was retarded, similar to the current study. In both studies, one with the hybrid and one with GH transgenic catfish, NaCl levels of 2.5-3.0 ppt were more beneficial for the growth of the slower growing genotypes at 0.0 ppt. The effect of salinity on growth rate of freshwater fishes appears to among

species,

and

is

affected

by

feed

consumption,

digestion,

f

vary

oo

utilization, and metabolic rate (Morgan and Iwama, 1991; El-Sayed, 2006;

pr

Lisboa et al., 2015). In the current study and several others, fish growth rate was higher in brackish water environments than seawater and freshwater

e-

environments (Küçük et al., 2013; Abass et al., 2017). However, other

Pr

research showed that fish growth rate was higher in freshwater environments than saltwater or seawater environments (Wang et al., 1997; Altinok and

al

Grizzle, 2001). Our results and those of Allen and Avault (1970), Lewis

rn

(1972), and Abass et al. (2017) indicated that the salinities of 0·85 to 4 ppt

freshwater

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enhanced growth rate of channel catfish. Additionally, the tolerance of fishes

to

different

concentrations

of salinity appears to be

dependent on species, strain, sex, size, genetics, life stage, adaptation time, and method and environmental factors (Morgan and Iwama, 1991; Varsamos et al. 2002; El-Sayed, 2006; Luz et al., 2008; Enayati et al., 2013; Abass et al., 2017). The current study and many others indicate that GH transgenesis can result in greatly increased growth rate in fish from 2 to an incredible 40 fold (Dunham, 2003; Devlin et al., 2004, 2006; Hobbs and Fletcher, 2008; Raven et al., 2008; Higgs et al., 2009; Leggatt et al., 2012; Tibbetts et al., 2013; 15

Journal Pre-proof Devlin et al., 2015). Dunham et al. (2002) reported that F1 and F2 rainbow trout GH cDNA transgenic common carp Cyprinus carpio grew 3% to 37% and 0% to 49% faster than their non-transgenic siblings, respectively, depending upon family. At one year of age, the transgenic Atlantic salmon Salmo salar possessing chinook salmon GH cDNA driven by the ocean pout antifreeze protein gene grew 2- to 6- fold larger than non-transgenic control

f

and the largest transgenic fish grew 13 times larger than the average non-

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transgenic (Du et al., 1992) and Oreochromis niloticus transgenic possessing

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chinook salmon GH cDNA driven by the ocean pout antifreeze protein grew 2.5 to 4 fold faster than non-transgenic siblings (Rahman et al., 1998, 2001;

e-

Rahman and Maclean, 1999; Caelers et al., 2005). The current study indicated

Pr

that the growth enhancement of GH transgenic catfish was seen at a variety of salt levels and the extent of that enhancement was variable. Pleiotropic effects

rn

genotypes or families.

al

on survival and condition factor were also observed and variable among GH

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Condition factor, growth and survival were not perfectly correlated, and genotype-environment interactions occurred for the overall phenotype. The growth and condition factor of rtMT-ccGH transgenic channel catfish both increased as salinity was increased to 2.5 ppt, and then both dropped as salinity increased further. Growth enhancement was less affected for opAFPccGH transgenic when salinity was raised to 2.5 ppt, and their condition factor was relatively stable at all salt levels. Responses of the control channel catfish were quite variable, and indicative of potentially large family or strain effects in regards to the response

to

increasing

NaCl.

The 16

non-transgenic

full-siblings

of

the

Journal Pre-proof transgenic individuals had small increases in growth at 2.5 ppt, whereas, the Kansas control responded with greater increases in body weight, and the albinos more than doubled their body weight at 2.5 ppt. All of the controls had severe mortality at 7.5 ppt, however, all but rtMT-ccGH controls had very high condition factors with that control and the transgenics having condition factors one-half to one-third that of the other three controls. The

f

surviving controls of these groups had very slow growth, but robust body

oo

shapes and condition.

controls

has

an

interesting

pr

This increased condition factor coupled with small size in most of the corollary

in

natural

saline

environments.

e-

Largemouth bass, Micropterus salmoides, in the brackish water area of the

Pr

Mobile Delta, Alabama USA, have slow growth rate and very high condition factors (Glover et al., 2013; DeVries et al., 2015). These fish migrate to and

al

tolerate different levels of salinity during different life sages, have differing

rn

physiological responses, alterations in feeding strategies, relatively short lives

Jo u

and appear to be genetically distinct. Similar alterations in blue catfish in this environment, specifically, the Tensaw River, are seen in this environment (Dunham and Smitherman, 1984). Interestingly, most of the control catfish in this study seem to have rapidly made similar adaptions in relative body shape, but the GH transgenics did not. The response by the controls could be partially related to epigenetics, which can cause major changes in days, and future study of epigenetic alterations might help explain some of the phenomenon seen in the current study. GH transgene increases a channel catfish’s and perhaps other fishes adaptation to a wider range of environmental conditions, salinity and extreme 17

Journal Pre-proof cold (Abass et al., 2017). Their performance was improved at 2.5 ppt, but controls with less growth potential benefited more from the higher salinity. However, at more extreme NaCl concentrations (7.5 ppt), survival of the transgenics was much greater than non-transgenic controls. However, their growth was impaired to the extent that commercial aquaculture would not be feasible. Older and larger sizes of fish should be evaluated as salinity

f

tolerance increases with size in other studies of ictalurid catfish. The variable

oo

responses of different GH and promoter combinations and the apparent

pr

family/strain variation hint that further breeding and selection efforts could improve performance in saline environments further and this should be Even if future performance would allow commercially feasible

e-

evaluated.

Pr

aquaculture at high saline conditions for freshwater species, brood stock would need to be spawned in areas with adequate salt water.

al

Multiple experiments indicate that inferior genotypes of catfish excel at

rn

2.5-3.0 NaCl. Thus, addition of salt can actually improve performance, but is

Jo u

likely not economically feasible. Low saline aquifers exist in West Alabama and catfish farmers have utilized this water to culture catfish and reduce disease problems. However, this practice has been reduced in frequency as farmers now believe that low saline water promotes toxic algae blooms (William Hemstreet, Auburn University, personal communication). Climate change may bring combinations of problems to solve. The current study shows that GH genetic engineering technology might be an initial step to assist in overcoming this future problem. However, the interactions in this type of environment are complex and further study is needed and warranted to make major impact. When these complexities are 18

Journal Pre-proof better understood, control and utilizatization of these variables coupled with GH transgenic meat technology could be highly beneficial to the aquaculture industry in the future to feed 9.6 billion people expected in the world by 2050. Before this can happen, concerns about risks of transgenic aquatic organisms, research on food safety and environmental risk; including the measurement of fitness traits such as predator avoidance and reproduction,

f

are needed to allow for informed decisions on the risk of using transgenic fish

oo

(Hallerman and Kapuscinski,1995; Dunham and Winn, 2014; Wakchaure et

pr

al., 2015; Leggatt et al., 2017). The increased tolerance of GH transgenic catfish could lead to the expansion of the geographic range of channel catfish that

would

be

negated

if

they

e-

however,

cannot

reproduce

in

these

Pr

environments. Although reproduction is unlikely in environments of 2 ppt and higher (Abass et al., 2016), reproduction of GH transgenic channel catfish at

al

high salinity should be evaluated. However, even if the conditions of

rn

reproduction are altered, risk can be prevented by total spawning control of

al., 2016).

Jo u

the fish with transgenic sterilization (Li et al., 2018) or gene editing (Qin et

FDA (2015) has approved triploid Atlantic salmon containing a chinook salmon GH transgene driven by the ocean pout antifreeze promoter (opAFP-GHc2) for commercial production for the first time for human consumption in the USA. This approval will lead to production of other transgenic GH meat for human consumption and may allow us to use this technology

to

Canada has

also

allow

future

approved

aquaculture the

to

consumption

adjust of

GH

to

climate

change.

transgenic

salmon

(Democracy Now, 2016; Waltz, 2017; Coghlan, 2017), and the first 4 tonnes 19

Journal Pre-proof of transgenic salmon have been marketed, sold and consumed. The results from the present study illustrate an example of how genetic engineering has the potential to help us adapt to environmental change.

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-p

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Journal Pre-proof Table 1 Mean (±SD) percent cumulative survival of different genetic groups of swim-up fry of

transgenic

hormone

channel catfish

(ccGH)

metallothionein

cDNA

promoter

(Ictalurus driven

(rtMT),

by

punctatus) the

transgenic

containing

rainbow

trout

channel

catfish

channel catfish

growth

Oncorhynchus

mykiss

(Ictalurus

punctatus)

containing channel catfish growth hormone (ccGH) cDNA driven by the ocean pout

Zoarces

americanus

N2

End of 1 St Week

End of 2 nd Week

End of 3 rd Week

of

Genotype1

End of 30 day

Jo

ur

na

lP

re

-p

ro

Salinity (ppt)

albino

channel

catfish

antifreeze throughout

protein study

promoter period

for 30 days.

30

in

(opAFP), different

channel

catfish,

concentrations

and

of NaCl

Journal Pre-proof

5

7.5

100.00±0.00

100.00±0.00

100.00±0.00

39

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

8

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

37

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

45

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

45

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

6

100.00±0.00

100.00±0.00

39

100.00±0.00

5

100.00±0.00

40

100.00±0.00

45

100.00±0.00

45

100.00±0.00

of

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00

100.00±0.00 a

83.33±28.87a

83.33±18.87a

83.33±18.87a

89.93±4.61a

82.41±4.80a

82.41±4.80a

82.41±4.80a

100.00±0.00a

83.33±18.87a

83.33±18.87a

83.33±18.87a

39

94.87±8.88a

87.18±11.75a

76.92±7.70 a

76.92±7.70a

45

95.33±3.85a

93.33±6.67a

88.89±10.18a

88.89±10.18a

45

66.66±5.77b

40.00±5.00b

40.00±5.00b

40.00±5.00b

6

100.00±0.00a

66.67±28.87a

66.67±28.87a

66.67±28.87a

39

66.67±11.75b

25.64±4.44b

20.51±4.45b

12.82±4.44b

5

66.67±28.87b

66.67±28.87a

66.67±28.87a

66.67±28.87a

40

65.38±17.63b

32.61±5.19b

15.20±7.97b

10.07±4.61b

40

-p

re

lP

na

5

ro

100.00±0.00

ur

2.5

6

6

Jo

0

rtMT-ccGH (T) rtMT-ccGH (C) opAFP ccGH (T) opAFP ccGH (C) Channel catfish Albino chann el catfish rtMT-ccGH (T) rtMT-ccGH (C) opAFP ccGH (T) opAFP ccGH (C) Channel catfish Albino chann el catfish rtMT-ccGH (T) rtMT-ccGH (C) opAFPccGH (T) opAFPccGH (C) Channel catfish Albino chann el catfish rtMT-ccGH (T) rtMT-ccGH (C) opAFPccGH (T) opAFP-

31

Journal Pre-proof ccGH (C) Channel catfish Albino chann el catfish

catfish mykiss

(T)

growth

22.22±3.85b

17.78±3.85b

45

26.66±5.77c

20.00±5.00b

6.67±1.53b

6.67±1.53b

=

channel

catfish,

Ictalurus

hormone

(ccGH)

cDNA

driven by the rainbow trout Oncorhynchus

metallothionein

sibling

non-transgenic

driven

by

promoter (control)

rainbow

trout

(rtMT), for

punctatus,

rtMT-ccGH

channel

catfish

metallothionein

(C)

transgenic

=

growth

promoter

channel

hormone

(rtMT),

for

channel

catfish

(ccGH)

fullcDNA

opAFP-ccGH

(T)

=

-p

the

37.78±3.85b

of

rtMT-ccGH

68.88±3.85b

ro

1

45

transgenic for channel catfish growth hormone (ccGH) cDNA driven

by

pout

ocean

opAFP-ccGH

(C)

=

Zoarces

americanus

antifreeze

protein promoter (opAFP),

channel catfish full-sibling non-transgenic (control) for

and

channel

lP

the

re

channel catfish

na

catfish growth hormone (ccGH) cDNA driven by the ocean pout antifreeze protein promoter (opAFP). Means that do not differ at the P = 0.05 are followed by the same (Duncan's

multiple

range

ur

superscript

test)

among

2

Jo

level of time and NaCl concentration. N=number of swim- up fry of ictalurid catfishes.

32

different

genetic

groups

at

fixed

Jo

ur

na

lP

re

-p

ro

of

Journal Pre-proof

33

Journal Pre-proof

Figure 1 Design of constructs, PCR strategy, and PCR analysis used in the gene transfer

study.

(A) rtMT-ccGH and

opAFP-ccGH plasmid

map.

(B) PCR strategy.

The position of different primers is indicated. (C) PCR analyses of plasmids DNA

construct

=

channel

driven

by

catfish the

(Ictalurus rainbow

punctatus)

trout

Oncorhynchus

hormone

(GH)

mykiss

metallothionein

-p

promo ter (rtMT).

growth

ro

rtMT-ccGH

of

with differe nt primers. cDNA

re

opAFP-ccGH = channel catfish growth hormone (GH) cDNA construct driven by the

Jo

ur

na

lP

ocean pout antifre eze protein Zoarces americanus promoter (opAF P).

34

of

Journal Pre-proof

ro

Figure 2 Mean (±SD) percent survival of different genetic groups of swim-up fry of transgenic

by

the

rainbow

trout

Oncorhynchus

mykiss

metallothionein promoter (rtMT),

re

driven

-p

channel catfish (Ictalurus punctatus) containing channel catfish growth hormone (ccGH) cDNA

transgenic channel catfish containing channel catfish growth hormone (ccGH) cDNA driven by

lP

the ocean pout Zoarces americanus antifreeze protein promoter (opAFP), channel catfish,

na

and albino channel catfish in different concentrations of NaCl at the end of the experiment. rtMT-ccGH (T) = channel catfish transgenic for channel catfish growth hormone (ccGH) cDNA

ur

driven by the rainbow trout metallothionein promoter (rtMT), rtMT-ccGH (C) = channel catfish

Jo

full-sibling non-transgenic (control) for channel catfish growth hormone (ccGH) cDNA driven by the rainbow trout metallothionein promoter (rtMT),

opAFP-ccGH (T) = channel catfish

transgenic for channel catfish growth hormone (ccGH) cDNA driven by the ocean pout antifreeze protein promoter (opAFP), and opAFP-ccGH (C) = channel catfish full-sibling nontransgenic (control) for channel catfish growth hormone (ccGH) cDNA driven by the ocean pout antifreeze protein promoter (opAFP). Means that do not differ at the P = 0.05 are followed by the same superscript (Duncan's multiple range test) among different genetic groups at fixed level of NaCl concentration.

35

Journal Pre-proof

Figure 3 Mean final body weight ± SD of

of

(g)

ro

different

-p

genetic

re

groups of swim-up fry of transgenic channel catfish (Ictalurus punctatus) containing channel catfish growth hormone (ccGH) cDNA driven by the rainbow trout Oncorhynchus mykiss containing channel catfish growth

lP

metallothionein promoter (rtMT), transgenic channel catfish

na

hormone (ccGH) cDNA driven by the ocean pout Zoarces americanus antifreeze protein promoter (opAFP), channel catfish, and albino channel catfish in different concentrations of

(T)

=

channel

catfish

transgenic

for

channel

catfish

growth

hormone

Jo

rtMT-ccGH

ur

NaCl at the end of the experiment.

(ccGH) cDNA driven by the rainbow trout metallothionein promoter (rtMT), rtMT-ccGH (C) =

channel

catfish

full-sibling

non-transgenic

(control)

for

channel

catfish

growth

hormone (ccGH) cDNA driven by the rainbow trout metallothionein promoter (rtMT), opAFP-ccGH

(T)

=

channel catfish

transgenic

for

channel catfish

growth

hormone

(ccGH) cDNA driven by the ocean pout antifreeze protein promoter (opAFP), and opAFP-ccGH (C) =

channel catfish full-sibling non-transgenic (control) for

channel

catfish growth hormone (ccGH) cDNA driven by the ocean pout antifreeze protein

36

Journal Pre-proof promoter (opAFP). Means that do not differ at the P = 0.05 are followed by the same superscript (Duncan's multiple range test) among different genetic groups at fixed level of NaCl

lP

re

-p

ro

of

concentration.

catfish,

Ictalurus

(ccGH)

cDNA

driven

growth

(rtMT), hormone

antifreeze

by

protein

the

channel catfish,

Jo

promoter

punctatus,

ur

channel

na

Figure 4 Comparison of growth enhancement in F1 of different genetic groups of swim-up fry of

(ccGH)

cDNA

promoter

transgenic

rainbow

trout

Ictalurus driven

(opAFP),

for

channel catfish

Oncorhynchus

punctatus,

by

the

channel catfish,

mykiss

transgenic

ocean

pout

and

growth

hormone

metallothionein

for channel catfish Zoarces

albino

americanus

channel catfish

at

different concentrations of NaCl. Fish are 44 days of age. a) rtMT-ccGH (T), b) opAFP- ccGH (T), c) channe l catfish (control), and d) albino channel catfish (contro l). rtMT-ccGH

(T)

=

channel

catfish

transgenic

for

channel

catfish

growth

hormone

(ccGH) cDNA driven by the rainbow trout metallothionein promoter (rtMT), and opAFP-

37

Journal Pre-proof ccGH (T) =

channel catfish transgenic for channel catfish growth hormone (ccGH)

Jo

ur

na

lP

re

-p

ro

of

cDNA driven by the ocean pout antifree ze protein promoter (opAFP).

38

ro

of

Journal Pre-proof

Figure 5 Mean condition factor ± SD of different genetic groups of swim-up fry of transgenic

by

the

rainbow

trout

Oncorhynchus

mykiss

metallothionein promoter (rtMT),

re

driven

-p

channel catfish (Ictalurus punctatus) containing channel catfish growth hormone (ccGH) cDNA

lP

transgenic channel catfish (Ictalurus punctatus) containing channel catfish growth hormone (ccGH) cDNA driven by the ocean pout Zoarces americanus antifreeze protein promoter

rtMT-ccGH

(T)

ur

end of the experiment.

and albino channel catfish in different concentrations of NaCl at the

na

(opAFP), channel catfish,

=

channel

catfish

transgenic

for

channel

catfish

growth

hormone

=

channel

Jo

(ccGH) cDNA driven by the rainbow trout metallothionein promoter (rtMT), rtMT-ccGH (C) catfish

full-sibling

non-transgenic

(control)

for

channel

catfish

growth

hormone (ccGH) cDNA driven by the rainbow trout metallothionein promoter (rtMT), opAFP-ccGH

(T)

=

channel catfish

transgenic

for

channel catfish

growth

hormone

(ccGH) cDNA driven by the ocean pout antifreeze protein promoter (opAFP), and opAFP-ccGH (C) =

channel catfish full-sibling non-transgenic (control) for

channel

catfish growth hormone (ccGH) cDNA driven by the ocean pout antifreeze protein promoter (opAFP). Means that do not differ at the P = 0.05 are followed by the same 39

Journal Pre-proof superscript (Duncan's multiple range test) among different genetic groups at fixed level of NaCl

Jo

ur

na

lP

re

-p

ro

of

concentration.

40

Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or per sonal relationships that could have appeared to influence the work reported in this paper.

Jo

ur

na

lP

re

-p

ro

of

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

41

Journal Pre-proof

-

Statement of Relevance Climate change may bring combinations of problems to solve. The current study shows that GH genetic engineering technology might be an initial step to assist in overcoming this future problem. Very

important

to

improve

salinity

tolerance

will

contribute

substantially to future utilization in brackishwater aquaculture to face

ur

na

lP

re

-p

ro

of

the shortage of freshwater for human usage in the future.

Jo

-

42

Journal Pre-proof

-

Highlights GH transgenic channel catfish had higher survival rate than their nontransgenic siblings and all other control channel catfish, and albino channel catfish at 7.5 ppt.

-

Massive

mortality occurred

for

different

genetic

groups

during

the

second

week at 7.5 ppt except for GH transgenic channel catfish. Swim-up

fry of channel catfish,

GH transgenic channel catfish, and albino

of

-

Condition

factor,

growth

and

survival were

not

perfectly

correlated,

ur

na

lP

re

-p

genoty pe- environ ment interaction s occurred for the overall phenotype.

Jo

-

ro

channel catfish had better growth in 2.5 ppt than 0 ppt.

43

and