larvae following cypermethrin exposure

Journal Pre-proof Phenotypic and trascriptomic changes in zebrafish (Danio rerio) embryos/larvae following cypermethrin exposure T. Sri Ranjani, Gopi Krishna Pitchika, K. Yedukondalu, Y. Gunavathi, T. Daveedu, S.B. Sainath, G.H. Philip, Jangampalli Adi Pradeepkiran PII:

S0045-6535(20)30341-6

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126148

Reference:

CHEM 126148

To appear in:

ECSN

Received Date: 9 December 2019 Revised Date:

6 February 2020

Accepted Date: 6 February 2020

Please cite this article as: Ranjani, T.S., Pitchika, G.K., Yedukondalu, K., Gunavathi, Y., Daveedu, T., Sainath, S.B., Philip, G.H., Pradeepkiran, J.A., Phenotypic and trascriptomic changes in zebrafish (Danio rerio) embryos/larvae following cypermethrin exposure, Chemosphere (2020), doi: https:// doi.org/10.1016/j.chemosphere.2020.126148. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Conceptualization: S.B. Sainath, G.H. Philip, Methodology: S.B. Sainath, G.H. Philip and Jangampalli Adi Pradeepkiran. Investigation:T. Sri Ranjani, Gopi Krishna Pitchika, K. Yedukondalu, Y. Gunavathi, T. Daveedu. Resources: S.B. Sainath, Data Curation: S.B. Sainath, T. Sri Ranjani, Gopi Krishna Pitchika, Jangampalli Adi Pradeepkiran. Writing Original Draft: S.B. Sainath, Gopi Krishna Pitchika, Writing- Reviewing and Editing: S.B. Sainath, Jangampalli Adi Pradeepkiran Supervision: S.B. Sainath, G.H. Philip.

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Phenotypic and trascriptomic changes in zebrafish (Danio rerio) embryos/larvae following

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cypermethrin exposure

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T. Sri Ranjani1,2$, Gopi Krishna Pitchika3$, K. Yedukondalu3, Y. Gunavathi3, T. Daveedu4,

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S.B. Sainath4*, G.H. Philip1* and Jangampalli Adi Pradeepkiran5,6*

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1

Department of Zoology, Sri Krishnadevaraya University, Anantapuramu-515003

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2

Department of Zoology, D.K. Govt. Degree College for Women (Autonomous), Dargamitta,

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Nellore-524003

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Department of Zoology, Vikrama Simhapuri University Post-Graduation Centre, Kavali-524201

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4

Department of Biotechnology, Vikrama Sihapuri University, Nellore-524320

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5

Department of Internal Medicine, Texas Tech University of Health Science Centre, Lubbock-

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79413, TX, USA.

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Department of Zoology, Sri Venkateswara University, Tirupati-517502, AP, India.

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Equal Contribution

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Corresponding authors:

Prof. G.H. Philip ([email protected])

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Dr. S.B. Sainath ([email protected])

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Dr. Jangampalli Adi Pradeepkiran

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([email protected])

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Abstract

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Cypermethrin is one of the widely used type-II pyrethroid and the indiscriminate use of this

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pesticide leads to life threatening effects and in particular showed developmental effects in

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sensitive populations such as children and pregnant woman. However, the molecular

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mechanisms underlying cypermethrin-induced development toxicity is not well defined. To

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address this gap, the present study was designed to investigate the phenotypic and transcriptomic

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(next generation RNA-Seq method) impact of cypermethrin in zebrafish embryos as a model

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system. Zebrafish embryos at two time points, 24 hours postfertilization (hpf) and 48 hpf were

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exposed to cypermethrin at a concentration of 10µg/L. Respective control groups were

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maintained. Cypermethrin induced both phenotypic and transcriptomic changes in zebrafish

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embryos at 48 hpf. The phenotypic anomalies such as delayed hatching rate, increased heartbeat

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rate and deformed axial spinal curvature in cypermethrin exposed zebrafish embryos at 48 hpf as

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compared to its respective controls. Transcriptomic analysis indicated that cypermethrin

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exposure altered genes associated with visual/eye development and gene functional profiling also

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revealed that cypermethrin stress over a period of 48 hrs disrupts phototransduction pathway in

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zebrafish embryos. Interestingly, cypermethrin exposure resulted in up regulation of only one

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gene, tnnt3b, fast muscle troponin isoform 3T in 24 hpf embryos as compared to its respective

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controls. The present model system, cypermethrin exposed zebrafish embryos elaborates the

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toxic consequences of cypermethrin exposure during developmental stages, especially in fishes.

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The present findings paves a way to understand the visual impairment in sensitive populations

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such as children exposed to cypermethrin during their embryonic period and further research is

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warranted.

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Keywords: Cypermethrin; development toxicity; Zebrafish; embryos; Transcriptomic analysis.

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Introduction

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Cypermethrin (CP) is one of the type II pyrethroids used to protect the economically

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important crops such as cotton, fruits and vegetables against wide range of insects of different

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arthropod classes, leipdoptera, hemiptera and coleopteran. Further, it is used in household

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purposes to eradicate cockroaches, lice and mosquitoes. CP is also used to control pests in

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various activities like wool processing and sheep dipping (USEPA, 1998), to control salmonids

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for sea lice (Ernst etal., 2001), in forestry (Torstensson et al., 1999), and also to control pests of

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cotton and soybean, moths etc., (Carriquiriborde et al.,2007). Therefore, the broad spectrum

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activity of CP attracted the attention of farmers and as a consequence its consumption increased

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globally. Moreover, lower toxicity to the mammals and birds and low persistence in the soil,

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makes the CP an important product of agriculture. However, due to repetitive usage of this

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pyrethroid, the non-target organisms such as humans and animals are exposed to CP and

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therefore, there is a much scope for the elevated risk of intoxication in non-target organisms via

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contaminated water (Vryzas et al., 2011; Singh et al., 2012).

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The toxic effects of CP has been reported in humans and experimental models and is

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mostly associated with nervous system disorders (Power and Sudakin, 2007). In humans, the

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toxic effects of CP include hypersensitivity reaction, reflex hyper excitability, tremors, throat and

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epigastric pain, nausea, headache, dizziness and fatigue (Aggarwal et al., 2015). Studies of

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Lessenger (1992) reported that five workers who inadvertently exposed to CP showed shortness

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of breath, nausea and headache. Further, fertility related problems and epileptic signs has been

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reported in humans exposed to CP (Condes-Lara et al., 1999). CP-induced cytotoxicity has also

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been reported in human lymphocytes (Charvarthi et al., 2007). In experimental models, multiple

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toxic effects of CP have been documented, such as neurotoxicity (Singh et al., 2012),

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reproductive toxicity (Pitchika et al., 2019), cardiac problems (Grewal et al., 2010),

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hepatotoxicity (El-Tawil and Abdel-Rahman, 2001; Grajeda-Cota et al., 2004; Sushma and

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Devasena, 2010) and nephrotoxicity (Grewal et al., 2010). Further, it has been shown that CP

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exposure interferes with sodium channels thereby negatively affect the neural signal transduction

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mechanisms (Wolansky et al., 2006; Wolansky and Harrill, 2008). The other toxic effects

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induced by CP include limb weakness, ataxia and muscular tremors to serious onset of

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convulsions, coma and respiratory depression followed by death of organism (Ullah et al., 2006).

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CP enters the aquatic ecosystem via agricultural run-off and thus, fishes are easily susceptible

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targets to CP. In fishes, exposure to CP causes abnormalities in behaviour, biochemical and

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haematological variables, inhibition of tissue antioxidant status and developmental toxicity,

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neurotoxicity, reprotoxicity and histological changes in the gills and intestine (Moore and

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Warming, 2001; Marino and Ronco, 2005; Prashanth and David, 2006; Carriquiriborde et al.,

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2007; Prashanth et al., 2011; Ullah et al., 2018).

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It is believed that the embryonic period is crtical for the development and differentiation

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of vital organs. Therefore, disturbances at this level may harm developing fetus, and

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consequently alters phenotypic changes later in life. Published reports have shown that the

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perinatal exposure to CP impairs reproductive development of female rats at their adulthood

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(Obinna and Agu, 2019). Prenatal and perinatal exposure to CP showed negative effects on

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eyelid opening, righting reflex acquisition, eye opening, pinna development and auditory startle

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reflex in experimental models, suggesting that the developing brain is one of the vulnerable

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targets of CP (Husain et al., 1992; Singh et al., 2015; Laugeray et al., 2017). Moreover, CP was

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detected in the plasma samples of workers in Pakistan (Khan et al., 2010) and in the urine of

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pregnant women, children and infants (Whyatt et al., 2002; Berkowitz et al., 2003; Lu et al.,

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2006; 2009). Most notably, CP-induced developmental toxicity in non-target organisms is of

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major concern, as the negative effects may persist from one generation to another.

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Danio rerio (zebrafish) is globally accepted alternative model for vertebrates as it possess

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exceptional portfolio of features such as short life cycle, ease of culture, prolific egg production

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with high fertilization as compared to traditional mice models, transparency during early life

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stages and the experiments designed in zebrafish embryos are believed to be pain-free and

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embryonic development is vulnerable to environmental stress (Zhou et al., 2009; Babcock et al.,

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2017; Shabnam and Philip, 2018). The other important features include, the genomic similarity

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between zebrafish and humans are at least 87% (Howe et al., 2013), and this high level of gene

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homology results in high conservation of signaling cascades between the zebrafish and humans

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(Chakraborty et al., 2016).

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In the present study, zebrafish embryos were exposed to low concentrations of

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cypermethrin i.e. 10 µg/L at two different time points, 24 hours post fertilization (hpf) and 48 hpf

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to evaluate the developmental toxicity by considering the a) phenotypic variables such as

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hatching efficiency, eye pigmentation, heartbeat, somitogenesis and axial spinal curvature, and

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b) transcriptomic analysis. By analyzing the results, this study attempts to assess the

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developmental toxicity of cypermethrin in zebrafish embryos.

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Materials and methods

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1.1.

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Chemicals Cypermethrin

(a-Cyano-(3-phenoxyphenyl)methyl

3-(2,2-dichlorovinyl)-2,2-

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dimethylcyclopropanecarboxylate; CAS Number: 52315-07-8) was obtained from Sigma-

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Aldrich (St Louis,MO, USA) (Fig. 1). In this study, molecular kits such as Trizol reagent (Sigma

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Aldrich, St Louis, MO, USA) and other molecular kits including first strand cDNA synthesis kit 5

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(iScriptTM, Bio-Rad, CA, USA), Taq polymerase with high fidelity Phusion mixture (Thermo-

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Scientifics, Waltham, USA), SYBR green master mix (Applied Biosystems, Warrington, UK)

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and Trueseq RNA library prep Kit (Illumina, San Diego, CA) were used. The molecular grade

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water used in this study was obtained from HiMedia (India). All other chemicals used in this

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study were of analytical grade.

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1.2.

Fish maintenance and breeding

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Adult zebrafish were purchased from a local pet shop (Nellore, Andhra Pradesh, India)

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and reared in the animal house facility available at Department of Zoology, VSUPG Centre,

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Kavali, Nellore District, AP, India for two generations. All the fishes were maintained in

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constant aerated 100L glass aquaria (50 fishes/tank) under defined laboratory conditions:

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dechlorinated tap water at 26 ± 20 C, continuous aeration to maintain dissolved oxygen levels

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between 7.5 to 8 µg/L and photoperiod of 12:12hrs light and dark cycles. Fish in stock were fed

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twice a day, with alternating diet of freshly hatched brine shrimp (Sanders Brine Shrimp Co,

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Utah) and dry flake food (Tetra, Germany). Male and female fishes were kept in breeding boxes

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(2:1 ratio) overnight prior to spawning. The breeding was finished at the beginning of the light

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cycle and the fertilized eggs were collected, washed twice with fresh water. The embryos that

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reached blastula stage were considered as normal (observed under microscope) and used for the

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current experiments. .

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1.3.

Experimental design

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Briefly, the fertilized eggs at 2 hours post-fertilization (hpf) were collected and

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transferred in to glass petridishes. Each dish (n=25) received 40 ml of filtered water containing

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10µg/L concentration of CP. The CP stock solution was prepared by dissolving 5 mg of CP in

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1.0 ml of absolute alcohol and stored at -200C. The stock solution (2 µl) was diluted with filtered

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water (1000 ml) to get working concentration i.e. 10 µg/L of CP whenever needed. In this study,

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we selected 10 µg/L of CP as a test dose based on our previous study, wherein we noticed CP

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exposure even at 10 µg/L induced spermatotoxicity in zebrafish (Pitchika et al., 2019). CP

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treated embryos were maintained in triplicates, up to 24 hpf (E24) and 48 hpf (E48). Respective

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solvent control groups (C24 and C48) were also maintained simultaneously which received

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alcohol alone. The embryonic development was monitored and the parameters such as mortality

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rate, hatching rate and malformations such as pericardial edema, pigmentation, and axial spinal

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curvature in the hatched larvae were recorded during the exposure period (Shabnam and Philip,

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2018). No significant effects were noticed in the selected phenotypic alterations such as mortality

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rate, hatching rate, heart beat rate and axial spinal curvature in zebrafish embryo/larvae exposed

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to solvent (2 µl of absolute alcohol diluted to 1 L of filtered water) as compared to controls at

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selected time points (data not shown).

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RNA isolation, reverse transcription and transcriptomic analysis

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The process of pipeline of transcriptomic analysis was illustrated in (Supplementary

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material: Fig. 1). Embryos from each group were pooled and homogenized using sterilized

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mortar and pestle in the presence of liquid nitrogen. Total RNA was isolated from the embryos

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using Qiagen mini RNeasy kit). After isolation of total RNA, the quality was analyzed

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spectrophotometrically and quantity of total RNA was performed using Qubit analysis (Life

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Technologies). Further, RNA electrophernogram and RNA integrity number (RIN) was

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determined using Bioanalyzer (Agilent Technologies). RIN values provide objective metric of

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total RNA quality ranging from 10 (high intact RNA) to 1 (degraded RNA). In this study, the

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RIN values for RNA isolated from zebrafish embryos (24 hrs)/larvae (48 hrs) were found to be

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8.5 for both control and experimentals. After completion of the quantitative and qualitative

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analysis, total RNA (1 µg) was reverse transcribed to cDNA libraries using Trueseq RNA library

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prep Kit (Illumina, San Diego, California). Library preparation was performed according to the

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manufacturer’s instructions. The qualified libraries were subjected to (Agilent Bioanalyzer

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2100), transcriptomic analysis on Illumina HiSeq 2000 platform using paired-end sequencing

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mode (2 x 100 bp) to match more accurately the reference genome sequence and improve

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sequence efficiency. Illumina-adapted library pools were prepared by a commercial service

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(Agrigenome Pvt. Labs Ltd., Hyderabad, Telangana State, India). Based on the quality of

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sequence reads, trimming was performed where necessary to retain only high quality sequence.

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In addition, the low-quality sequence reads were excluded from the analysis. The pre-processed

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reads were aligned to the zebrafish genome downloaded from Ensembl database

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(ftp://ftp.ensembl.org/pub/release-94/fasta/danio_rerio/dna/Danio_rerio.GRCz11.dna.toplevel.

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fa.gz). The alignment was performed using Hisat2 program (version 2.0.5) with default

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parameters. After alignment, the reads with reference gene model, the aligned reads were used

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for estimating expression of the genes and transcripts, using cufflinks program (version 2.2.1).

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More than 90% of total reads of all samples passed >= 30 Phred score (Supplementary material:

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Table 1). The differential expression of gene (DEGs) analysis was performed using cuffdiff

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program of cufflinks package with default settings. The genes were considered differentially

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expressed only if they attain q-value <= 0.05. The functional enrichment analysis of DEGs in

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terms of Gene ontology (biological process, cellular components and molecular functions) was

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analyzed using The Database for Annotation, Vsiualization and Integrated Discovery (DAVID;

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Version: v6.8) (Huang et al., 2008), Protein Analysis Through Evolutionary Relationships

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(Panther; Version: 14.1) (Mi et al., 2019) and g:profiler (Raudvere et al., 2019) databases which

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facilitate high-throughput analysis.

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1.4.

Validation of transcriptomic data using qPCR

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To validate the differential expression of genes obtained from transcriptomic analysis, the

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expression of five selected differentially expressed genes were determined with qPCR. The

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selected genes were tnnt3b, pax6a, pax2a, sox2, and six3b in CP exposed zebrafish embryos at

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selected time points. Briefly, total RNA was isolated using the Trizol plus purification system

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(Invitrogen, Carlsbad, USA) and the purity of RNA was analyzed spectrophotometrically

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(Model: Jasco V-750; Mary’s CourtEaston, MD 2160) and by agarose gel electrophoresis. The

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quantity of purified RNA was determined using NanoDrop-2000 spectrophometer (Thermo-

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Fisher Scientific). The first strand cDNA synthesis was performed as per the manufacturer’s

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instructions of iscriptTM cDNA synthesis kit (Biorad, India) using 1 µg of total RNA. The reverse

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transcribed cDNA was used for expression of selected genes using quantitative Real-Time PCR

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(qPCR; Applied Biosystems). The primer pairs (Supplemenatry material: Table 2) for respective

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genes

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(http://www.ncbi.nlm.nih.gov/tools/primer-blast). Prior to the use of primer pairs, the efficiency

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of primers were analyzed by standard curves from a dilution series. The efficiency of primers

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was found to be >90%. The qRT-PCR assay was carried out using SYBRTM green master mix

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(Thermo-Fisher Scientific) and analyzed on step-one real time PCR system (Agilent

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technologies, Stratagene, Mx3005P). All samples were run in triplicates, including a negative

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control. The mean Ct values were determined from the triplicates. The obtained Ct values were

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used for quantification of normalized expression according to the 2-∆∆Ct method (Schmittgen and

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Livak, 2008) using reference gene beta actin. The data was expressed as relative mRNA

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expression after normalization for each sample in CP exposed and the control groups.

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1.5.

were

designed

using

the

Statistical analysis 9

NCBI-Primer

Blast

tool

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For qRT-PCR experiments, one treatment group comprised of 4 wells of zebrafish

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embryos. Three such samples were used for the interpretation of data. The data were represented

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as mean ± SD. and statistically analyzed using non-parametric student’s t-test for comparison

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between two groups using GraphPad Prism 5.01 (GraphPad Software Inc., La Jolla, CA, USA).

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The P values < 0.05 were considered as statistically significant.

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The DEGs were subjected to three standalone gene enrichment analysis tools, DAVID

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version 6.7 (https://david.ncifcrf.gov/), PANTHER (www.pantherdb.org/) and g:profiler

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(https://biit.cs.ut.ee › gprofiler). For DAVID, the statistical parameters were Benjamini multiple

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test correction. For PANTHER database, the statistical parameters were Bonferroni correction

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for multiple testing set at p<0.05. For g:profiler, the statistical parameters were Benjamini

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Hochberg False discovery rate (FDR) with a threshold of 0.05. The DEGs between the control

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and experimental groups were also represented via Venn diagram (Heberle et al., 2015).

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2.

Results

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2.1.

Developmental toxicities

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No mortality was observed in any of the control and experimental groups. CP (10 µg/L)

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exposure showed phenotypic malformations such as delayed hatching rate, increased heartbeat

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rate and deformed axial spinal curvature in zebrafish larvae over a period of 48 hpf as compared

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to C48 group (Fig. 2).

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3.2.

Gene enrichment analysis

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Of the 32518 genes annotated, 21092 genes were mapped using Panther and DAVID

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databases. The gene enrichment analysis indicated that out of 21092 genes, 20922, 14323 and

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14105 and 7079 genes were categorized to biological process, cellular component, molecular

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function and KEGG pathways, respectively (supplementary material: Tables 3 and 4).

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Transcriptomic analysis identified the profile of differentially expressed genes (DEGs) in

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zebrafish embryos after 24 hpf and 48 hpf exposure to CP at a dose of 10µg/L. A Venn diagram

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demonstrated the over lapped and specific genes between the groups C24 versus C48, C24

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versus E24 and C48 versus E48 (Fig. 3).

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In total, 59 genes (up-regulation: 50 and down-regulation: 9; Supplementary material:

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Table 5) were found to be significantly altered in expression during the development as the

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zebrafish embryos progress from 24 hpf stage to 48 hpf . The gene enrichment analysis indicated

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that out of 50 genes, a total of 42 genes were mapped using panther database and out of which 26

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genes were categorized under molecular function, 34 genes were categorized under biological

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process and 28 genes were categorized under cellular component. Most of the genes under the

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gene enrichment term molecular function (13 out of 26 genes), biological process (10 out of 34

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genes) and cellular component (10 out of 28 genes) fall under the sub-categories binding (GO:

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0005488), cellular process (GO: 0009987) and cell (GO: 0005623), respectively. Further, the

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functional profiling of genes under biological process indicated that most of the genes were

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related to vision/ocular development (Supplementary material: csv file1). With respect to the

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down regulated genes between C24 versus C48, only 37.5% (i.e. 3 out of 8) genes were enriched

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under the category biological process. The functional profiling of top four biological processes of

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down regulated genes between C24 versus C48 groups were anterior/posterior axis speciation,

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regionalization, axis speciation and somitogenesis (Supplementary material: csv file1). KEGG

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pathway analysis showed that up-regulated genes (cldn11a: claudin11a and myhz1.1: myosin,

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heavy polypeptide 1.1 skeletal muscle) in C48 group were predominantly enriched in tight

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junction related pathway (DAVID analysis; p value: 5.9E-2 and benjamini value: 2.2E-1:

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Supplementary material: Fig. 2).

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On the other hand, only one gene i.e. tnnt3b was found to be differentially expressed in

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E24 group as compared to C24 group. Interestingly, a gradual up-regulation of tnnt3b was

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observed as the embryos progress from 24 hpf stage to 48 hpf stage and while such response was

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not observed in the expression of tnnt3b in E48 group as compared to E24 group (Table 1).

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In total, 43 genes (Table 1) were found to be differentially expressed in E48 group as

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compared to C48 group and out of which 24 genes were up regulated and 19 genes were down

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regulated. Interestingly, majority of the differentially expressed genes in E48 group were found

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to be different from that of genes that were observed between C24 versus C48 group. The gene

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enrichment analysis indicated that out of 24 up regulated genes, a total of 23 genes were mapped

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using panther database and out of which 19 genes were categorized under molecular function, 33

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genes were categorized under biological process and 14 genes were categorized under cellular

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component. Most of the genes under the gene enrichment term molecular function (7 out of 19

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genes), biological process (8 out of 33 genes) and cellular component (6 out of 14 genes) fall

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under sub-categories transporter regulator activity (binding (GO: 0005488), metabolic process

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(GO: 0008152) and organelle (GO: 0043226), respectively. Further, the functional profiling of

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genes under biological process indicated that most of the differentially expressed genes under CP

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stress over a period of 48 hpf were related to vision/ocular development (Supplementary

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material: csv file 3). With respect to the down regulated genes between C48 versus E48, there

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were 16, 16 and 9 functional hits under the categories biological process, molecular function and

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cellular component, respectively (Supplementary material: csv file 4). Most of the genes under

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the gene enrichment terms biological process (6 out of 16 genes), and molecular function (6 out

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of 16 genes) fall under the subcategories multicellular organismal process and bidning,

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respectively. Further, functional profiling of down regulated genes in E48 group using g: profiler

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database indicated that cypermetrhin exposure disrupts phototransduction pathway in zebrafish

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larvae and the altered genes were gnat1 (Guanine nucleotide binding protein alpha transducing

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activity polypeptide 1) and pde6a (phosphodiesterase 6A, cyclic GMP specific, rod alpha)

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(DAVID analysis; p value: 1.9E-2; Benjamini value: 9.0E-2; Fold enrichment value: 79.5; EASE

281

value: 0.1) (Figure 4).

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qPCR studies

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In order to authenticate the differential genes detected with RNA-seq, five genes were

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selected randomly from DEGs belonging to the visual/ocular development and one gene related

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to troponinT and subjected to transcriptional validation. The results indicated that expression

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patterns of selected genes by qRT-PCR were in concurrence with those by RNA-seq (Table 2).

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Discussion

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The present findings demonstrated that CP exposure at 10 µg/L induced significant

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noticeable signs of developmental toxicity in zebrafish embryos as evidenced by phenotypic

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abnormalities such as delay in hatching process, increased heartbeat rate, and deformed axial

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spinal curvature. Our results support the studies of Sathya et al. (2014) and Shabnam and Philip

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(2018). Hatching process is one of the developmental indicators, as this process is linked to

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biochemical and physical aspects. In order to hatch, the outer chorion layer of the egg is digested

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by hatching enzyme and to accomplish this task, normal tail, notochord and axial spinal cord are

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important prerequisite. A delay in hatching process in zebrafish embryos which was observed in

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this study might be associated with the CP-induced disturbances at the level of structural and

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functional integrity of tail and/or notochord (Richterova et al., 2015; Liu et al., 2016). An

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increase in the pericardial edema observed in this study might be linked to the increased

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heartbeat rate associated with slow blood flow in CP exposed zebrafish embryos (Xu et al., 2010;

13

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Sathya et al., 2014). Notochord is a transient structure that functions to provide support to

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vertebrae and spine during embryogenesis (Zeng et al., 2018). Thus, malformed axial spinal

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curvature which was observed in this study might reflect notochord abnormalities in zebrafish

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embryos exposed to CP (Shabnam and Philip, 2018). In this study, the mortality rate was below

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1% in control zebrafish embryos/larvae as required for early life stage test validity (Kimmel et

305

al., 1995).

306

In the present study, high through-put data analysis using RNA-Seq was employed to

307

understand the moelcular basis of CP-induced toxicity in zebrafish embryos (24 hpf)/larvae (48

308

hpf). To our surprise, Tnnt3b (fast muscle troponin isoform) was found to be the only one gene

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that was altered in E24 group relative to their respective controls. While the expression levels of

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tnnt3b in E48 group was reduced as compared to E24 group. In contrast, the expression levels of

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tnnt3b mRNA increases as the zebrafish embryos progress from 24 hpf to 48 hpf under normal

312

conditions. Tnnt3b is predominant isoform of troponin T, in an adult zebrafish and found at the

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highest levels within immature hearts and serves as an indicator for the switch between

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fetal/neonatal and the adult heart (Stefancsik et al., 2003; Hsiao et al., 2003). Earlier studies have

315

shown that exposure of zebrafish embryos to sodium metam, an agricultural dithiocarbamate

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pesticide altered myogenesis associated genes including tnnt3b (Tilton et al., 2008). Knock-

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down studies conducted by Ferrante et al. (2011) have shown that tnnt3b also plays a key role in

318

the troponin T activity and eventually stabilizes sarcomere functions. From the findings, we

319

propose that the detection of tnnt3b (confirmed by qPCR studies) in zebrafish embryos at 24 hpf

320

stage might reflect early transcriptional indicator during CP-induced developmental toxicity. We

321

hypothesize that the altered expression of tnnt3b mRNA observed in CP-exposed zebrafish

14

322

embryos (24 hpf) might reflect disrupted cardiac muscular functions. Further studies are

323

warranted to support this notion.

324

At 48 hpf, many genes were found to be differentially expressed in controls and

325

experimental groups. In the current study, CP stress predominantly altered the genes related to

326

visual/eye development. It is well documented that during embryogenesis, visual system is

327

believed to be one of the vulnerable targets to environmental pollutants, as they can able to attain

328

ability to enter and accumulate in the eyes, followed by genomic and proteomic changes of

329

visual system and eventually disorganize and disturb visually mediated behavior (Xu et al., 2017;

330

Chen et al., 2018; Liu et al., 2018). Published reports indicated that CP exposure significantly

331

reduced eye size in fish embryos (Shi et al., 2011; Dawar et al., 2016).

332

Developmental exposure to CP altered genes related to retinal pigment epithelium (RPE),

333

and integral structural components of eye in zebrafish larvae at 48 hpf. It is believed that the

334

thickness (six3b and sox2) and differentiation (pax genes) of RPE from neural retina is pivotal

335

for the development of visual system in vertebrates as it is involved in the protection of light-

336

receptive cells (Baumer et al., 2003; Yasuo et al., 2009). The differentiation of RPE is precisely

337

regulated by microphthalmia-associated transcription factor in association with two genes pax6a

338

and pax2a and thus, down regulation of pax6a and pax2a (confirmed by qPCR data) might

339

suggest improper differentiation of RPE from neural retina in E48 group (Baumer et al., 2003).

340

Published reports also indicated that over expression of six3b and sox2 during xenopus and avian

341

embryogenesis, respectively negatively affect RPE morphogenesis and cellular diferntiation

342

(Bernier et al., 2000; Yasuo et al., 2009). Studies of Babich et al. (2019) demonstrated that

343

arsenic (500 ppb) exposure over a period of 48 hpf caused a significant reduction in the thickness

15

344

of RPE and attributed to the down regulation of pax genes and up regulation of six3b and sox2,

345

respectively in zebrafish larvae relative to controls.

346

Fatty acid binding protein 11b (fabp11b) is a paralog of zebrafish fabp11a and play a key

347

role in fatty acid metabolism of developing eyes (Karanth et al., 2008). In this study,

348

transcriptomic analysis revealed that as the zebrafish embryo (24 hpf) transforms to larvae (48

349

hpf), the expression of fabp11b increases and in constrast, down regulation of fabp11b in

350

zebrafish larvae was found under CP stress relative to controls. This might indicate that the

351

developmental exposure of zebrafish larvae to CP primarily targets

352

metabolism in the ocular system. .

fabp11b of fatty acid

353

Previous studies highlighted that exposure of zebrafish embryos/larvae to pollutants

354

altered gene expression levels of crystallins associated with impairment of eye development

355

(Chen et al., 2016; Xu et al., 2016; Shi et al., 2019). Crystallins are integral structural

356

components of eyes to regulate the transparency and function of lens and cornea (Vihtelic et al.,

357

1999). Among the crystallins, gammaM crystallins (crygm) are abundantly expressed during

358

early life stages of zebrafish and are essential for underwater vision (Easter and Nicola, 1996).

359

Based on the transcriptomic data, under normal conditions as the zebrafish embryos progress

360

from 24 hpf to 48 hpf, the gamma crystallin genes such as crygmd1, crygmd2, crygmd4,

361

crygmd5, crygmd6, crygmd7, crygmd9, crygmd11, crygmd12, crygmd17, crygmd20, crygmd21

362

and crygmxl2 were found to be over expressed. Interestingly, over expression of crygmd3

363

associated with absence of expression of crygmd1, crygmd6, crygmd17, crygmd20, crygmd21

364

and crygmxl2 were found in E48 group relative to controls. Therefore, alterations in the

365

expression of gamma crystallins under CP stress can be used as one of the indicators of visual

366

toxicity. Other studies also demeonstrated that zebrafish larvae exposed to gold nanoparticles

16

367

(Kim et al., 2013), polycyclic aromatic hydrocarbons, phenanthrene (Huang et al., 2013) and

368

alkaloid cyclopamine (Stenkamp and Frey, 2003) also resulted in the impairment of ocular

369

development.

370

Zebrafish embryos/larvae are widely used to assess the chemical-induced toxicity at the

371

level of developmental processes as they comprise of evolutionary conserved structures and their

372

dynamic response to chemical stimulus (Vesterlund et al., 2011). Zebrafish embryos/larvae under

373

CP stress were subjected to transcriptomic analysis to understand the toxic effects of CP on

374

developmental process at the molecular level. In this study, we validated few genes using qPCR

375

studies and many more genes must be validated via qPCR and western blotting analysis. This is

376

one of the limitations of this study. Despite of this limitation, the present study provides valuable

377

information about the CP-induced birth defects.

378

In conclusion, CP exposure is one of the public health concerns and most notably, it has

379

been found in the urine of pregnant women. In this study for the first time we demonstrated the

380

developmental toxic effects of CP zebrafish embryos/larvae as model systems. Research

381

investigating how CP affect zebrafish development is more translatable to human biology

382

(Parng, 2005). Herein, we sought to link the developmental toxicological outcome(s) of CP (10

383

µg/L) at molecular level in zebrafish embryos/larvae using transriptomic analysis. The major

384

outcome of this study was two fold: a) exposure to CP causes phenotypic alterations such as

385

hatching rate, heartbeat rate and axial spinal curvature and b) transcriptomic analysis revealed

386

early indicators of CP-induced developmental toxicity in zebrafish embryos/larvae at the

387

molecular level. Based on the results, it can be concluded that CP developmental exposure

388

caused interference with cardiac muscle formation at 24 hpf, while altered genes related to ocular

17

389

development at 48 hpf. The developmental toxic effect of CP on zebrafish embryo was

390

illustrated in figure 5. The results of this study may pose a developmental hazard to mammals.

391

Acknowledgements

392

The authors thank the Head, Dept. of Zoology and Dept. of Biotechnology for supporting

393

the space to maintain animals and also providing instruments.

394

Conflict of interest

395 396

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27

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Table 1:

Effect of cypermethrin stress on zebrafish embryos (24 hrs)/larvae (48 hrs) transcriptome

599 600

Gene Symbol

Gene Name

Ensembl ID

Log fold change

Time point: 24 hrs zebrafish embryos exposed to cypermethrin over a period of 24 hrs as compared to unexposed embryos (24 hrs) Up-Regulated genes Tnnt3b

Fast muscle troponin T isoform TnnT3b

ENSDARG00000068457

5.15

Down-Regulated genes: No gene were differentially down-regulated Time point: 48 hrs zebrafish embryos as they progress from 24 to 48 hrs under cypermethrin stress Up-Regulated genes Crx

Cone-rod homeobox;crx;ortholog

ENSDARG00000011989 4.12

Cryaa

Alpha A crystallin;cryaa;ortholog

ENSDARG00000053502 3.28

cryba1l1

Beta A1-2-crystallin;cryba1l1;ortholog

ENSDARG00000032929 6.14

cryba2a

Beta A2-crystallin;cryba2a;ortholog

ENSDARG00000030349 6.36 ENSDARG00000007576

crybb1l1

Crystallin, beta B1,-like 1;crybb1l1;ortholog

ENSDARG00000016793

crybb1l2

Crystallin, beta B1,-like 2;crybb1l2;ortholog

crygm2d3

NA

ENSDARG00000088823 6.95

dnase1l1l

Deoxyribonuclease;dnase1l1l;ortholog

ENSDARG00000023861 3.54

foxq2

Forkhead box Q2;foxq2;ortholog

ENSDARG00000071394 3.12

GRIK2

NA

ENSDARG00000113771 2.97

krt15

Keratin 15;krt15;ortholog

ENSDARG00000036840 3.11 ENSDARG00000007715 4.28

Lgsn

Lengsin, lens protein with glutamine synthetase domain

neurod2

Neurogenic differentiation factor 2

ENSDARG00000016854 3.16

6.32 6.21

28

Neuronal PAS domain-containing protein 4A

ENSDARG00000055752 2.18

npas4a

ENSDARG00000045677 2.33

opn1sw1

Opsin-1, short-wave-sensitive 1;opn1sw1;ortholog

ENSDARG00000076978 1.89

Pmchl

Pro-melanin concentrating hormone-like protein

si:ch211196c10.13

Si:ch211-196c10.13;si:ch211196c10.13;ortholog

ENSDARG00000096756 2.96

Si:ch211-255g12.6

ENSDARG00000094310 2.36

six3b

Homeobox protein Six6;six3b;ortholog

ENSDARG00000054879 3.12

six7

Homeobox protein Six7;six7;ortholog

ENSDARG00000070107 3.25

slc4a8

Anion exchange protein;slc4a8;ortholog

ENSDARG00000015531 3.91

sox2

Transcription factor Sox-2;sox2;ortholog

ENSDARG00000070913 2.28

Tnmd

Tenomodulin;tnmd;ortholog

ENSDARG00000052615 4.13

si:ch211255g12.6

zgc:172323 Zgc:172323;zgc:172323;ortholog

ENSDARG00000053201 4.29

Down-regulated genes ENSDARG00000019763 -3.68

acp5a

Tartrate-resistant acid phosphatase type 5;acp5a;ortholog

crygm2d11

Crystallin, gamma M2d8

ENSDARG00000069827 -3.97

crygm2d12

Crystallin, gamma M2d12

ENSDARG00000069801 -4.29

crygm2d2

Crystallin, gamma M2d2

ENSDARG00000086917 -5.29

crygm2d4

NA

ENSDARG00000087164 -5.02 ENSDARG00000069792 -5.32

crygm2d5

Crystallin gamma EM2-5 (Crystallin, gamma M2d5)

ENSDARG00000076572 -3.98

crygm2d7

Crystallin gamma EM2-7 (Crystallin, gamma M2d7)

crygm2d9

Crystallin, gamma M2d9

ENSDARG00000115701 -4.26 ENSDARG00000002311 -3.25

fabp11b

Adipocyte fatty acid-binding protein (Fabp11b protein)

29

ENSDARG00000044199 -3.67

gnat1

Guanine nucleotide-binding protein (G protein), alpha transducing activity polypeptide 1 (Rod transducin alpha subunit)

Gnsb

N-acetylglucosamine-6-sulfatase

ENSDARG00000098296 -3.54 ENSDARG00000102722 -4.29

itih3b

Inter-alpha-trypsin inhibitor heavy chain 3b

ENSDARG00000017441 -6.25

mylz3

Fast skeletal muscle myosin light polypeptide3

pax2a

Paired box protein Pax-2a;pax2a;ortholog

ENSDARG00000028148 -2.87

pax6a

Paired box protein Pax-6;pax6a;ortholog

ENSDARG00000103379 -2.69

pde6a

Phosphodiesterase;pde6a;ortholog

ENSDARG00000000380 -3.98

slc25a1b

Slc25a1 solute carrier family 25

ENSDARG00000076381 -6.39

slc39a5

Solute carrier family 39 (zinc transporter)

ENSDARG00000079525 -1.98

601 602

30

603

Table 2:

Differentially expressed genes in zebrafish embryos/larvae confirmed by qPCR

604

Gene symbol

Fold change (RNA-Seq)

Fold Change (qPCR)c

tnnt3b

5.15a

6.29

pax6a

-2.87b

-3.08

pax2a

-2.69b

-2.97

sox2

2.28b

2.68

six3b

3.12b

2.39

605 606 607 608 609 610 611

a

Fold change refers to gene expression in the 24 hpf cypermethrin-treated samples compared with its respective control samples. b Fold change refers to gene expression in the 48 hpf cypermethrin-treated samples compared with its respective control samples. c p<0.05 by qPCR (n=3).

31

Figure Legends

612

613

Figure 1:

Chemical structure of cypermethrin

614

Figure 2:

Effect of cypermethrin (10 µg/L) on Mortality rate (a: 24 hpf and 48 hpf),

615

hatching rate (b: 48 hpf), heartbeat rate (c: 48 hpf) and axial spinal curvature (d

616

and e: 48 hpf)

617 618

Figure 3:

The venn diagram represents the overlap between the significantly differentially expressed genes in the zebrafish embryos treated with or without cypermethrin

619 620 621

Figure 4:

KEGG

pathway analysis

of

down

regulated

genes

involved

in

the

622

phototransduction cascade under dark and light conditions. Genes encoding for

623

gnat1 (Guanine nucleotide binding protein alpha transducing activity polypeptide

624

1) and pde6a (phosphodiesterase 6A, cyclic GMP specific, rod alpha) in 48 hpf

625

zebrafish larvae exposed to cypermethrin during development were shown in red

626

boxes.

627 628

Figure 5:

Schematic illustration of developmental effects of cypermethrin on zebrafish

629

embryos/larvae. Exposure of zebrafish embryos (24 hrs) to cypermethrin shows

630

up-regulation of tnnt3b gene (* indicates up-regulation). Exposure of zebrafish

631

larvae (48 hrs) to cypermethrin shows phenotypic and transcriptomic changes.

632

Cypermethrin exposure alters genes related to lens, fatty acid metabolism and

633

retinal pigment epithelium development (RPE). During normal development of

634

eye, the gamma crystallin genes such as crygmd1, crygmd2, crygmd4, crygmd5, 32

635

crygmd6, crygmd7, crygmd9, crygmd11, crygmd12, crygmd17, crygmd20,

636

crygmd21 and crygmxl2 were expressed in the lens of zebrafish larvae. However,

637

over expression of crygmd3 associated with absence of expression of crygmd1,

638

crygmd6, crygmd17, crygmd20, crygmd21 and crygmxl2 was observed in lens of

639

zebrafish larvae exposed to cypermethrin. With regard to fatty acid metabolism,

640

fabp11b gene was down regulated (** indicates down regulation) in the ocular

641

system of zebrafish larvae exposed to cypermethrin over controls. With regard to

642

RPE, pax6a** and pax2a** were down regulated and six3b* and sox2* were up

643

regulated in zebrafish larvae exposed to cypermethrin over controls.

33

Highlights
Zebrafish embryos exposed to cypermethrin (10µgL-1) over a period of 24 hrs alters only one gene (tnnt3b) related to cardiac muscle development
Zebrafish larvae exposed to cypermethrin (10µgL-1) over a period of 48 hrs induced phenotypic malformations
Zebrafish larvae exposed to cypermethrin (10µgL-1) over a period of 48 hrs induced ocular developmental toxicity
The present study provides valuable information about the early transcriptome indicators in zebrafish embryos/larvae following cypermethrin exposure