Journal of Comparative Physiology B https://doi.org/10.1007/s00360-018-1151-8

ORIGINAL PAPER

Effects of the recombinant crustacean hyperglycemic hormones rCHH-B1 and rCHH-B2 on the osmo-ionic regulation of the shrimp Litopenaeus vannamei exposed to acute salinity stress Laura Camacho‑Jiménez1,2   · Fernando Díaz1   · Edna Sánchez‑Castrejón1   · Elizabeth Ponce‑Rivas1  Received: 10 October 2017 / Revised: 23 February 2018 / Accepted: 3 March 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract The Pacific white shrimp Litopenaeus vannamei is a euryhaline organism that copes with salinity fluctuations in the environment; therefore, its osmotic and ionic regulation abilities are vital. Osmoregulation may be controlled by the crustacean hyperglycemic hormone (CHH), a neuropeptide mainly expressed in the eyestalks. In L. vannamei, CHH-B1 and CHH-B2 are CHH isoforms isolated from the eyestalks whose expression is influenced by environmental salinity. It has been suggested that they are involved in the response to salinity stress. To clarify this, we investigated the effect of the recombinant peptides, rCHH-B1 and rCHH-B2, on the osmo-ionic regulation of shrimp acutely exposed to different salinity conditions (8, 26 and 45‰). Both rCHHs promoted differential effects on the osmoregulatory capacity (OC) and the ionoregulatory capacity (IC) for hemolymph ­Na+ and ­Cl− during iso-osmotic (26‰) and hyper-osmotic (45‰) transfers. These changes were linked to the changes observed in ­Na+/K+ ATPase and carbonic anhydrase gene expression in gills, especially under high salinity conditions, suggesting that the hormones may regulate the expression of these genes. Glucose and protein levels measured during acute salinity transfer suggest their roles as sources of metabolic energy for osmotic regulation or as organic osmolytes. These results taken together suggest that both the CHH-B1 and CHH-B2 peptides are important regulators of the physiological response of L. vannamei to acute salinity fluctuations. Keywords  Shrimp · Crustacean hyperglycemic hormone · Salinity · Osmo-ionic regulation · Carbonic anhydrase · Na+-K+ATPase

Introduction Salinity is one of the most influential environmental factors affecting the physiological performance of aquatic organisms (Péqueux 1995; Romano and Zeng 2012; Li et al. 2014; Thabet et al. 2017). Salinity fluctuations could affect shrimp in estuaries and in pond cultures as a result of evaporation, Communicated by G. Heldmaier. * Elizabeth Ponce‑Rivas [email protected] 1



Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada, Carretera Ensenada‑Tijuana #3918, CP 22860 Ensenada, Baja California, Mexico



Present Address: Centro de Investigación en Alimentación y Desarrollo (CIAD), A.C., Carretera Ejido a la Victoria Km 0.6, CP 83304 Hermosillo, Sonora, Mexico

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freshwater inflow or rainfall (Gucic et al. 2013; Zhang et al. 2016). The capacity of penaeids to accomplish metabolic and physiological adjustments to cope with wide changes of environmental salinity is a prime factor for their growth and survival (Díaz et al. 2001; Rosas et al. 2001; Valdez et al. 2008). The Pacific white shrimp Litopenaeus vannamei is able to grow in inland, coastal and oceanic waters due to its wide tolerance to salinity (1–40‰), which is one reason for the commercial success of this species in aquaculture worldwide (Menz and Blake 1980; Saoud et al. 2003; Kumaran et al. 2017). Osmoregulation is often vital for aquatic organisms, allowing the adjustment of the osmotic conditions of their fluids and tissues with respect to those in the environment (Péqueux 1995). Even though many classic euryhaline crustacean models (e.g., crabs) behave as osmoconformers, not osmoregulators, at higher salinities (e.g., > 27‰) (Mantel and Farmer 1983; Henry 2005), penaeid shrimp such as L. vannamei is, in general, osmotic and ionic conformers

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only within a narrow range of 25–27‰ (Díaz et al. 2001). Penaeid shrimp typically behaves as hyper-osmoregulators at low salinities, actively uptaking salts from the surrounding water, and at high salinities behaves as hypo-osmoregulators, excreting ions to the environment (Castille and Lawrence 1981; Díaz et al. 2001; Charmantier et al. 2009). However, the osmoregulatory capacity and osmotic concentration of hemolymph decrease with increasing L. vannamei shrimp size (Wickins 1976; Gong et al. 2004). Because of this, larvae can survive an acute transfer to freshwater, whereas juveniles are usually exposed to gradual salinity changes, of about 5% per day (Castille and Lawrence 1981; Rosas et al. 2001). Despite these studies, a little is known about the actual physiological mechanisms of salinity adaptation in L. vannamei (Roy et al. 2007). The ability of crustaceans to regulate hemolymph osmotic and ionic concentrations is believed to depend on the presence of a number of ion transport proteins and transportrelated enzymes in the gills, such as the ion transporter (pump) complex N ­ a+-K+-ATPase (NKA) and the enzyme carbonic anhydrase (CA) (Péqueux et al. 1984; Lovett et al. 2006; Freire et al. 2008; Charmantier et al. 2009; Henry et al. 2012). In euryhaline crabs, such as Callinectes sapidus and Eriocheir sinensis, NKA activity is concentrated in the posterior gill epithelium. However, in the freshwater, crayfish Pacifastacus leniusculus and in the hyper/hypo regulating white shrimp L. vannamei CA induction occurred in both anterior and posterior gills in response to low salinity exposure (Wheatly and Henry 1987; Palacios et al. 2004; Roy et al. 2007). The presence or absence of anterior/posterior gill differentiation has suggested a relationship between the environmental demands placed on the animals and its ability to maintain a proper salt and water balance (Wheatly and Henry 1987). The NKA pump is a transmembrane oligomeric enzyme composed of three subunits: α, β and γ (Blanco and Mercer 1998). The catalytic α-subunit carries out the ATPdependent ­Na+ uptake in exchange for ­K+ (or N ­ H4+) (Lucu and Towle 2003; Geering 2008), and its gene expression has been shown to be salinity responsive in diverse crustaceans (Charmantier et al. 2009; Thabet et al. 2017), including penaeid shrimp. For example, in Penaeus monodon, the NKA α-subunit expression in gills increased considerably after acclimatization to low (3‰) and high (55‰) salinities by ~ 34- and ~ 16-fold, respectively (Shekhar et al. 2013, 2014). Moreover, the acute transference of L. vannamei from a salinity nearly iso-osmotic (30‰) to low salinities (1, 7.5, 15‰) slightly induced the α-subunit expression in gills by ~ 1–2-fold after 6 h (Sun et al. 2011). In addition to NKA, CA is one of the most studied osmoregulatory genes and proteins in crustaceans. It catalyzes the reversible conversion of ­CO2 + H2O to ­HCO3− + H+, which serves as a mechanism for ­CO2 excretion and acid–base regulation, but also

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­ l− uptake (Burnett et al. provides counter ions for N ­ a+ and C 1985; Henry 1988; Henry et al. 2003). In crustaceans, it has been suggested that at least three CA isoforms are expressed in gills, with sub-cellular location in the cytoplasm, basolateral membrane and mitochondria (Henry 2005). The expression of CA genes in euryhaline crustaceans usually increases in the osmoregulatory gills, principally following salinity transfers from higher to low salinities. However, studies in euryhaline decapods have suggested that the cytosolic CA is more sensitive to salinity since in these organisms CA is up-regulated by at least 15-fold (Henry 1988, 2001, 2005; Henry et al. 2003). For example, a 100-fold increase of cytoplasmic CA expression was observed in C. sapidus (Serrano et al. 2007) and Carcinus maenas (Serrano and Henry 2008) after high to low salinity transfers. In L. vannamei, cytoplasmic CA mRNA expression was up-regulated to ~ 3-fold 24 h post-transfer from 30‰ to either 20 or 35‰ salinity (Liu et al. 2015). Studies in decapods have demonstrated that osmo-ionic regulation is controlled by neuroendocrine molecules originating in the eyestalks (Kato and Kamemoto 1969; Charmantier et al. 1984; Mantel 1985; Freire et al. 1995). In the eyestalks, there is the x-organ–sinus gland complex (XO­–SG), the main neuroendocrine structure that synthetizes and releases the crustacean hyperglycemic hormones (CHHs), a group of neuropeptides ubiquitous among crustaceans and having pleiotropic effects (Webster et al. 2012). The CHHs have been shown to participate in the control of the osmolyte and water concentrations of hemolymph and tissues in diverse species (Charmantier-Daures et al. 1994; Chung et al. 1999; Spanings-Pierrot et al. 2000; Serrano et al. 2003; Prymaczok et al. 2016). The injection of CHH has been demonstrated to partially restore the OC of eyestalk-ablated Homarus americanus in diluted medium (Charmantier-Daures et al. 1994), and the ion concentrations in the hemolymph of eyestalk-ablated crayfish Astacus leptodactylus (Serrano et al. 2003). These effects seem to depend on changes in the influx of ions across the gills (Spanings-Pierrot et al. 2000) by controlling the NKA activity, as has been shown in L. vannamei (Liu et al. 2014). In contrast, the eyestalk ablation of the euryhaline crabs C. maenas and C. sapidus potentiated the inductor effect of the low salinity transference on cytosolic CA expression and activity, suggesting its inhibition by an unknown eyestalk neuropeptide (Henry 2006; Henry and Borst 2006; Henry and Campoverde 2006). In L. vannamei, CHH-B1 and CHH-B2 are CHH isoforms produced in the XO­–SG by alternative splicing of the chhB gene (Lago-Lestón et al. 2007). CHH-B1 (GenBank: AAN86056) is a CHH-like (CHH-L) peptide with a free amino acid at the C terminus, whereas CHH-B2 (GenBank: AAN86057) has the typical amide moiety found at the C terminus of the CHH peptides (Lago-Lestón et al. 2007;

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Ma et al. 2010). The mRNAs encoding both peptides seem to fluctuate in response to the molt cycle, temperature and salinity (Lago-Lestón et al. 2007; Tiu et al. 2007). The transcript expression of CHH-B1 and CHH-B2 in the eyestalks increases under extreme low and high salinity conditions (Lago-Lestón et al. 2007). Recombinant CHH-B1 (rCHHB1) has been shown to increase the glucose levels in the hemolymph (Sánchez-Castrejón et al. 2008; Camacho-Jiménez et al. 2015) and to increase the OC of L. vannamei under hyper-osmotic salinity (Camacho-Jiménez et al. 2017a). In contrast, the recombinant CHH-B2 (rCHH-B2) has been shown to decrease the OC of shrimp under hyper-osmotic conditions (Camacho-Jiménez et al. 2017b). This evidence suggests the involvement of these CHH/CHH-L peptides in the hydromineral balance and the osmotic stress response in L. vannamei. Hyperglycemia stimulated by CHHs is a rapid response to diverse stressors, including salinity changes (Lorenzon 2005; Shinji et al. 2012). Thus, the CHH and CHH-L hormones could be important to rapidly adjust osmoregulatory mechanisms in response to salinity fluctuations. However, there is inconclusive evidence of whether these neuropeptides directly coordinate the specific mechanisms of ion transport in crustaceans, or exert their effect through metabolic activities, by supplying the energy necessary for these processes. The rCHH-B1 and rCHH-B2 hormones may have a modulatory role on the NKA and CA activity in white shrimp upon salinity stress that may occur at the gene expression level, since both enzymes are salinity sensitive. In this sense, the present study investigates the effect of rCHH-B1 and rCHH-B2 on metabolic, osmotic and molecular responses related to the osmo-ionic regulation of juvenile L. vannamei shrimp after a short-term (acute) exposure to hypo- or hyper-osmotic stress conditions.

Materials and methods Animals The L. vannamei shrimp used in the experiments were acquired as post-larvae (PL) from the commercial farm Acuacultura Mahr in La Paz, México. PL were grown in reservoirs (2000 l) filled with aerated seawater (SW) (35‰ and 28 ± 1 °C) until they reached the juvenile stage. For the experiments, 306 juvenile shrimp (7.3 ± 1.5 g) were transferred to individual containers (3.5  l) submerged inside reservoirs (200 l) with a continuous flow of aerated water (26 ± 1 °C) and 26‰ salinity, which is the isoosmotic point reported for juvenile L. vannamei shrimp (Diaz et al. 2001). The iso-osmotic salinity was achieved by mixing SW (35‰) with freshwater (FW) using a valve system, and was continuously monitored with a portable

refractometer. Daily, debris and feces were removed from the reservoirs. The animals were fed twice a day with a commercial shrimp feed (35% protein). Shrimp were acclimated to these conditions for 10 days before the experiments.

Recombinants CHH‑B1 and CHH‑B2 The expression of rCHH-B1 and rCHH-B2, having amino acid sequences identical to the native peptides from L. vannamei XO­– SG, in the methylotrophic yeast Pichia pastoris (strain X-33), as well as their purification by reversed-phase high-performance liquid chromatography (RP-HPLC) were performed as reported previously (Camacho-Jiménez et al. 2015). Concisely, colonies of X-33 carrying either pPICZαA-CHH-B1a or pPICZαA-CHH-B2b integrated in its genome, expressing the rCHH-B1 and rCHH-B2 peptides, respectively, were grown for 18 h in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose) with zeocin (100 µg/ ml) (250 rpm, 30 °C). The cultures were transferred to 500 ml of BMGY (buffered glycerol-complex) medium (1% yeast extract, 2% peptone, 0.67% yeast nitrogen base (YNB), 4 µg/mLD-biotin, 100 mM potassium phosphate buffer pH 6.0, 1% glycerol) and incubated until the cell density reached an ­OD600 ~ 4. The cells from 100 ml of BMGY cultures were harvested by centrifugation (3000g, room temperature, 5  min) and re-suspended in 20  mL of BMMY (buffered methanol-complex) medium (1% yeast extract, 2% peptone, 0.67% YNB, 4 µg/ml d-biotin, 100 mM potassium buffer pH 6.0) with 2% of methanol (v/v) and then incubated for 24 h (250 rpm, 30 °C). Fresh methanol was added every 12 h to sustain the induction. After the induction, the culture supernatants were collected (11,000g, 4 °C, 5 min), the proteins were precipitated with 50% ammonium sulfate (w/v), and then dialyzed against phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM N ­ a2HPO4, 1.8 mM ­KH2PO4, 0.05%, pH 7.4). The rCHHs were purified by RP-HPLC on a C18 column ­( TSKgel ® Octadecyl-4PW 4.6 mm × 150 mm, Tosoh, Tokyo, Japan) with an isocratic step of 5 min with 0.12% trifluoroacetic acid (TFA), followed by a gradient of 0–55% acetonitrile with 0.1% TFA at a flow rate of 1 ml/ min, over 40 min. Purification of rCHH-B1 and rCHHB2 was confirmed by tricine sodium dodecylsulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) (12.5%) (Fig. 1). The purified rCHHs were quantified by the bicinchoninic acid method, using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The peptides were stored at − 80 °C until the salinity stress assay.

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Fig. 1  Tricine-SDS-PAGE analysis of purified rCHH-B1 and rCHHB2. (M), precision plus protein all blue standards (Bio-Rad), (B1) rCHH-B1, (B2) rCHH-B2. The arrows indicate the bands corresponding to purified recombinant hormones

Acute salinity stress assay The experiment was done using only shrimp in intermolt acclimated as described in the “animals” section. The intermolt stage of each animal was determined as previously described (Camacho-Jiménez et al. 2015). Shrimp in intermolt were fasted for 24 h prior to the assay. Groups of 81 shrimp were injected through the arthrodial membrane, either with 2 µg of rCHH-B1 or rCHH-B2 dissolved in 50 µl of PBS using a 1-ml sterile hypodermic syringe (31 G). As negative control, 81 animals were injected with PBS. Immediately, 27 animals of each treatment and control were left under iso-osmotic conditions (26‰) (control salinity), or were transferred to either hypo- (8‰) or hyper-osmotic salinity (45‰). Low and high salinities were achieved either by diluting SW with FW, or by adding natural sea salt to SW, respectively. Also, groups of 27 un-injected shrimp were transferred to 8 and 45‰ salinity to monitor the stress due to the manipulation and salinity change. There were three sampling times (1, 3 and 6 h), and at each one, shrimp from each experimental condition were sampled (n = 9). Additionally, un-injected shrimp (n = 9) were taken from the acclimation reservoirs (26‰) and were used to determine the initial levels (0 h) for the measured responses.

Hemolymph analyses Hemolymph was collected from the thoracoabdominal membrane previously dried with absorbent paper with a 1-ml sterile hypodermic syringe (27 G). For osmotic pressure (OP) measurement, 10 µl of fresh hemolymph were

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analyzed in a VAPRO 5520 vapor pressure osmometer (Wescor, Logan, UT, USA). The water OP from the reservoirs was also measured. The OC was calculated as the difference of the hemolymph OP minus OP of medium at a given salinity (Lignot et al. 1997). The hemocyanin (Hc) content in the hemolymph was measured from a diluted sample (1:10) in distilled water inside a 10-mm quartz cuvette (1 ml, 1-cm path length). The absorbance was read at 340 nm in a Spectronic 200 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the Hc concentration was calculated using an extinction coefficient of E = 17.26, calculated based on the minimal functional subunit (74 kDa) of Hc from crustaceans (Hagerman 1983). The remaining hemolymph was divided in two parts: one diluted in anticoagulant solution (1:2) (450  mM NaCl, 10  mM KCl, 10  mM EDTA·Na 2, 10 mM HEPES, pH 7.3, 850 mmol/kg) (Vargas-Albores et al. 1993), and the other with heparin ammonium salt (1:3) (Sigma–Aldrich, St. Louis, MO, USA) (0.8 mg/ml, pH 7.3). The samples were centrifuged (800g, 4 °C, 5 min) and the plasma fraction was recovered. The glucose and total proteins were measured from plasma in anticoagulant solution using the glucose (oxidase) reagent set (Pointe Scientific, Canton, MI, USA) and the Protein Assay kit (BioRad, Hercules, CA, USA), respectively. Ion concentrations were determined from the heparinized plasma. The N ­ a+ was measured with the sodium color reagent (Stanbio, Boerne, TX, USA) and the C ­ l− using the chloride reagent set (Pointe Scientific, Canton, MI, USA). The concentration of each ion in the water reservoirs was also measured. The ionoregulatory capacity (IC) for ­Na+ and ­Cl− was calculated as the difference in ion concentrations between the hemolymph and the external medium at a given salinity.

Total RNA extraction and cDNA synthesis After hemolymph sampling, a posterior gill of the eighth pair was dissected from each shrimp in every experimental condition (n = 9) and was pooled in groups of three gills per experimental condition (n = 3) to yield ~ 50 mg of tissue. The tissue was immediately treated with RNA stabilizing solution (25 mM sodium citrate, 10 mM EDTA, 70% ammonium sulfate (w/v), pH 5.2) and then frozen at − 80 °C. The total RNA from preserved tissue was extracted and treated with DNase I using the Direct-zol™ RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). The purity, integrity and quantity of the RNA were analyzed by electrophoresis in TAE/ formamide agarose gels according to Masek et al. (2005), and also by the ratio A260/280 nm using a spectrophotometer ­NanoDrop™ 2000 (Thermo Scientific, Wilmington, DE, USA). The first strand cDNA was synthetized from 1 µg of total RNA using the ­SuperScript® III Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA) and

Journal of Comparative Physiology B

Oligo (dT)20 primer by following the manufacturer’s instructions. The cDNA samples were stored at − 20 °C.

The plasmids were stored at − 20 °C before gene expression analysis.

Cloning

Real‑time quantitative PCR (RT‑qPCR) analysis

To get a standard to use during the qPCR assays the CA and NKA cDNA fragments were cloned into the ­pCR™ 2.1TOPO® vector, according to the manufacturer’s protocol. Specific primers for the amplification of a 137 bp carbonic anhydrase cDNA fragment by polymerase chain reaction (PCR) were designed from the sequence of L. vannamei CA (GenBank No: HM626273.1) (LvACI_F, 5′-AGC​AAT​GAA​ ATC​CTA​CCA​TCC-3′; LvACI_R, 5′-ACT​CTT​CCT​CCA​ TCT​TGT​CC-3′). This sequence has a 99.6% identity to the cDNA of cytoplasmic CA from L. vannamei reported by Liu et al. (2015). Primers to amplify a 122 bp ­Na+-K+-ATPase α-subunit cDNA fragment (NKA) were designed by Li et al. (2009) (LvαATP_F, 5′-AGC​AAG​GCC​ATC​AAC​GAT​CT-3′; LvαATP_R, 5′-GCC​CAC​TGC​ACA​ATC​ACA​AT-3′). Each PCR mixture (12.5 µl) contained 1X colorless ­GoTaq® reaction buffer (Promega, Madison, WI, USA), 4 mM M ­ gCl2, 0.4 mM dNTP, 0.4 µM of each primer, 1.25 U of G ­ oTaq® DNA polymerase, and 0.25 µl of cDNA. The reaction conditions consisted of an initial denaturation step at 95 °C for 2 min, followed by 30 cycles at 95 °C for 30 s, 60 °C for 1 min and 72 °C for 25 s, and a final extension step at 72 °C for 5 min. The PCR products cloned into the p­ CR™ 2.1-TOPO® vector were transformed into chemically competent Escherichia coli strain DH5α, according to the ­TOPO® TA ­Cloning® Kit instructions (Invitrogen, Life Technologies). Transformants were screened for antibiotic resistance as follows. Blue–white colonies found on Luria–Bertani (LB) agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) plates containing ampicillin (0.1 mg/ml) and X-gal (40 mg/ml) after being incubated overnight at 37 °C were selected and evaluated by PCR with the specific primers. A positive colony for each product was transferred to 3 ml of LB medium with ampicillin and grown overnight (37 °C, 250 rpm). Plasmids were purified from the cultures using the ­PureLink® HQ mini plasmid purification kit (Invitrogen, Life Technologies). The insertion of CA and NKA fragments into the vectors was confirmed by sequencing using M13 Forward primer (5′-TGT​AAA​ACG​ACG​GCC​AGT​-3′) (SeqxCel, San Diego, CA, USA). The plasmid concentration was measured using ­NanoDrop™ 2000 (Thermo Scientific) and the exact number of copies in the stocks was calculated by the following equation (Whelan et al. 2003):

The effects of rCHHs on the CA and NKA expression in the posterior gills of L. vannamei were analyzed by real-time quantitative PCR (RT-qPCR). The reactions for each gene (10 µl) were prepared using the Power ­SYBR® Green PCR Mix (Applied Biosystems, Life Technologies), mixed with 25 ng of cDNA and 0.1 µM of each specific primer (Applied Biosystems, Life Technologies, Foster, CA, USA) in a ­StepOnePlus™ Real Time PCR System (Applied Biosystems, Life Technologies). Reaction conditions consisted of a hot start step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 s and 60 °C for 1 min. No-template control was used to detect PCR contamination. A standard curve for each gene was included per run to determine the absolute quantity of mRNA in each sample (expressed as copies of transcript per ng of cDNA) from a linear regression line (R2 ≥ 0.99). Standard curves were constructed with dilutions of the plasmids carrying the specific fragments (­ 107–102 copies) using StepOne Software version 2.3 (Applied Biosystems, Life Technologies). Each experimental sample, as well as the standard curve and the negative control were analyzed in triplicate per run. At the end of each run, a melting curve analysis was performed to ensure that only the specific product was produced. The efficiency of each primer set was calculated from the background-corrected raw fluorescence data using LinRegPCR Software version 2016.0 (Ruijter et al. 2009), which were of 93.5 and 91.6% for CA and NKA, respectively.

Plasmid (copies/ng) = [6.022 × 1023 (copy/mol) × lasmid amount (ng)]∕plasmid + insert length (bp) × 660 (g/mol) (1) × 1 × 109

Statistical analyses The data were analyzed by the Kruskal–Wallis non-parametric test followed by a Dunn’s multiple comparisons post hoc test or a Mann–Whitney rank sum test to assess the differences with respect to the pre-injection control (0 h), as well as between the experimental groups in each sampling time. The data were expressed as median ± 95% confidence interval (CI). A p < 0.05 was considered as statistically significant. The statistical analyses and plots were done using the SigmaStat version 3.5 (Systat Software, San Jose, CA, USA) and SigmaPlot version 10.0 (Systat Software), respectively.

Results Effect of rCHHs on osmoregulatory capacity (OC) The time-course effects of the injection of rCHH-B1 and rCHH-B2 on the OC of shrimp exposed to different salinities are shown in Fig. 2. The initial (0 h) OC levels of the

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Fig. 2  Time-course effect of rCHH-B1 and rCHH-B2 on the osmoregulatory capacity (OC) of shrimp exposed to 8, 26 and 45‰ salinity. ND, no data. Data are expressed as median ± 95% CI (n = 9).

Asterisks indicate significant differences (p < 0.05) compared to the pre-injection group (0  h). Different letters indicate statistical differences (p < 0.05) between groups per sampling time (h)

un-injected shrimp under iso-osmotic conditions (26‰) were close to 0 (− 15.00 ± 10.93 mmol/kg), as their OP values were similar to the environmental level. At this salinity, rCHH-B2 caused a small, but significant decrease in the OC levels 6 h post-injection (− 45.00 ± 33.52 mmol/ kg) in comparison to the initial time (p = 0.016, Mann–Whitney test) and PBS (21.00 ± 20.80 mmol/kg) (p = 0.002, Mann–Whitney test) controls. The shrimp exposed to hypo-osmotic salinity (8‰) were hyper-osmotic with respect to the external medium, showing an OC significantly higher (H = 24.994, df = 4, p < 0.001, Kruskal–Wallis test) than the initial control. However, no differences (p > 0.05, Dunn’s test) were observed between treatments and controls 1 h after injection, and no data were collected at later sampling times due to the death of shrimp before reaching 3 h after the salinity change. The animals exposed to hyper-osmotic salinity (45‰) behaved as hyporegulators, with an OC lower (H = 28.952, df = 4, p < 0.001, Kruskal–Wallis test) than the initial control from the first hour. Only the rCHH-B1 injection modified the hypoosmoregulatory strength of shrimp by ~ 13% with respect to both un-injected (− 456.50 ± 21.23 mmol/kg) (p = 0.002, Mann–Whitney test) and PBS (− 450.50 ± 28.05 mmol/kg) (p = 0.018, Mann–Whitney test) controls. No differences (p > 0.05, Dunn’s test) were observed with respect to the controls at later sampling times.

promoted a significant hyperglycemia (p < 0.05, Dunn’s test) 1 h post-injection, regardless of the salinity condition. The highest increase was observed with the rCHH-B2 injection at 26‰ salinity, being ~ 1.5-fold higher in comparison to the PBS control (29.96 ± 3.96 mg/dl) (p < 0.001, Mann–Whitney test). Interestingly, a similar effect (p < 0.001, Mann–Whitney test) was observed with the two peptides in shrimp exposed to acute hypo- and hyper-osmotic salinities. The hyperglycemic effect decreased after 3 h post-injection for both hormones in shrimp exposed to 26 and 45‰.

Effect of rCHHs on the hemolymph glucose The initial hemolymph glucose level in shrimp acclimated at 26‰ was 13.55 ± 0.94  mg/dl (Fig.  3). Both rCHHs

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Effect of rCHHs on the hemolymph proteins The initial levels (0 h) of total proteins (Fig. 4a) and Hc (Fig. 4b) obtained in hemolymph were 127.12 ± 5.97 mg/ ml and 1.54 ± 0.21 mmol/ml, respectively. The injection of rCHH-B1 and rCHH-B2 into shrimp under iso-osmotic conditions caused an increase in the total protein levels 6 h after injection over the initial values (H = 67.987, df = 3 p < 0.001, Kruskal–Wallis test). The maximum level of proteins was observed with the rCHH-B2 injection, being ~ 23% higher than the level observed with the injection of the PBS control (190.19 ± 6.35 mg/ml) (p < 0.001, Mann–Whitney test). No significant changes (p > 0.05, Kruskal–Wallis test) were observed in the Hc values of iso-osmotic shrimp compared to the initial level. All shrimp acutely exposed to 8‰ salinity showed a reduction (H = 71.316, df = 4, p < 0.001, Kruskal–Wallis) in the total protein levels 1 h post-transfer, with no significant variations (H = 1.070, df = 4, p = 0.899, Kruskal–Wallis) in the Hc levels. Total protein and Hc levels of shrimp acutely exposed to 45‰ salinity decreased

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Fig. 3  Time-course effect of rCHH-B1 and rCHH-B2 on the hemolymph glucose of shrimp exposed to 8, 26 and 45‰ salinity. ND no data. Data are expressed as median ± 95% CI (n = 9). Asterisks indi-

cate significant differences (p < 0.05) compared to the pre-injection group (0 h). Different letters indicate statistical differences (p < 0.05) between groups per sampling time (h)

significantly (p < 0.05, Dunn’s test) at 6 h after injection with both rCHH peptides compared to the controls, with no differences (p = 0.051, Mann–Whitney test) between their effects. Total protein levels in animals injected with hormones decreased up to ~ twofold at 6 h post-transfer to 45‰ salinity in comparison with the un-injected (136. 93 ± 16.47 mg/ml) and PBS (130.26 ± 52.72 mg/ml) controls, respectively; while the Hc levels decreased by ~ 25 and ~ 32% with respect to the un-injected (1.28 ± 0.19 mmol/l) and PBS (1.14 ± 0.17 mmol/l) controls, respectively. No significant differences (p = 0.480, Mann–Whitney test) were observed in the Hc levels between the two peptides under these conditions.

p < 0.001, Kruskal–Wallis test) in comparison with the initial IC control, indicating that animals were hypo-regulating the hemolymph ions. A decrease (p < 0.001, Mann–Whitney test) in the IC levels for N ­ a+ was observed 1 h after injection of rCHH-B1 in comparison to the un-injected (− 357.50 ± 24.85 mEq/l) and PBS (− 334.90 ± 8.97 mEq/l) controls by ~ 20 and ~ 14.5%, respectively. The rCHH-B2 injection gradually reduced (p < 0.05, Dunn’s test) the IC levels for N ­ a+ reaching the lowest values at 6 h post-transfer, showing ~ 13% reduction in comparison with the un-injected (− 272.17 ± 13.36 mEq/l) (p = 0.003, Mann–Whitney test) and PBS (− 271.50 ± 18.40 mEq/l) (p < 0.001) controls. The IC levels for ­Cl− were reduced 6 h after rCHH-B2 injection by ~ 25 and ~ 22% in comparison with the un-injected (− 260.08 ± 11.30 mEq/l) (p = 0.003, Mann–Whitney test) and PBS (− 247.91 ± 11.35 mEq/l) (p < 0.001, Mann–Whitney test) controls, respectively.

Effect of rCHHs on the ionoregulatory capacity (IC) The IC levels for ­Na+ (Fig. 5a) and ­Cl− (Fig. 5b) in the 0  h control at 26‰ salinity were 87.48 ± 8.40 and − 19.74 ± 11.19  mEq/l, respectively. The recombinant neuropeptides did not elicit significant changes (p > 0.05, Dunn’s test) in the IC for both ions with respect to controls at any sampling time. Shrimp acutely exposed to 8‰ salinity showed a higher (H = 63.990, df = 4, p < 0.001, Kruskal–Wallis test) IC level for ­Cl− in comparison with the initial control, which is in agreement to the hyper-regulatory pattern expected for shrimp exposed to low salinities. However, no differences (p > 0.05, Dunn’s test) were observed between treatments and controls at that salinity. When shrimp were acutely transferred from 26 to 45‰ salinity, the IC levels decreased for both ­Na+ (H = 86.132, df = 4, p < 0.001, Kruskal–Wallis test) and ­Cl− (H = 59.751, df = 4,

Effect of rCHHs on the carbonic anhydrase and ­Na+‑K+‑ATPase expression The results for CA and NKA mRNA expression in posterior gills of L. vannamei are shown in Fig. 6a, b, respectively. Under iso-osmotic conditions, the initial CA expression in the un-injected shrimp was 3.85 ± 0.49 × 103 transcripts per ng of cDNA. This expression increased slightly, but significantly at 6 h post-injection of rCHH-B1 (p = 0.008, Mann–Whitney test). However, no differences were observed with respect to the PBS control (7.22 ± 0.15 × 103 transcripts per ng of cDNA) (p = 0.683, Mann–Whitney test). The basal NKA expression levels in un-injected shrimp at iso-osmotic conditions were

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Fig. 4  Time-course effects of rCHH-B1 and rCHH-B2 on the total proteins (a) and hemocyanin (b) concentrations in the hemolymph of shrimp exposed to 8, 26 and 45‰ salinities. ND no data. Data are expressed as median ± 95% CI (n = 9). Asterisks indicate significant

differences (p < 0.05) compared to the pre-injection group (0 h). Different letters indicate statistical differences (p < 0.05) between groups per sampling time (h)

9.21 ± 0.26 × 103 transcripts per ng of cDNA. The NKA transcripts were significantly (p < 0.05, Dunn’s test) up-regulated 3 h after rCHH-B1 and rCHH-B2 injection at 26‰ salinity. Under these conditions, rCHH-B2 injection significantly increased NKA expression levels by ~ 37.5% as compared with the PBS control (p = 0.037, Mann–Whitney test). At 8‰ salinity, only rCHH-B2 promoted a slight, but significant increase (p = 0.016, Mann–Whitney test) in CA expression at 1 h posttransfer with respect to the initial control, which was not significantly different (p = 1.00, Mann–Whitney test) from the un-injected levels (5.43 ± 0.44 × 103 copies per ng of cDNA). The NKA expression levels of shrimp acutely exposed to this hypo-osmotic salinity were significantly lower (H = 21.615,

df = 4, p < 0.001, Kruskal–Wallis test) than the initial control. However, no significant differences (p > 0.05, Dunn’s test) were observed among treatments and controls 1 h after injection of the rCHH peptides. Under hyper-osmotic conditions, the expression of both genes was significantly up-regulated (p < 0.05, Dunn’s test) at 1 h post-injection of the rCHH peptides, in contrast to controls. The CA expression was increased (p > 0.05, Dunn’s test) with both hormones at least ~ 28% over the PBS levels (7.07 ± 0.74 × 103 copies per ng of cDNA). The highest effect on the NKA expression was observed 1 h after rCHH-B1 injection, which promoted a ~ 1.5-fold increase over the PBS control (16.66 ± 1.25 × 103 copies per ng of cDNA) (p = 0.002, Mann–Whitney test). At 3–6 h after rCHH-B1

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Fig. 5  Time-course effects of rCHH-B1 and rCHH-B2 on the ionoregulatory capacity for ­Na+ (a) and ­Cl− (b) of shrimp exposed to 8, 26 and 45‰ salinity. ND no data. Data are expressed as median ± 95% CI (n = 9). Asterisks indicate significant differences

(p < 0.05) compared to the pre-injection group (0 h). Different letters indicate statistical differences (p < 0.05) between groups per sampling time (h)

injection, the expression levels of both genes were similar or significantly decreased in comparison to the initial control.

2017), information about the specific mechanisms used by these hormones to carry out these processes is still scarce. In this work, we used recombinant versions of CHH-B1 and CHH-B2 isoforms to study their effects on diverse metabolic, physiological and molecular responses related to osmo-ionic regulation in L. vannamei after a sudden transfer to extreme salinity conditions. To assess the osmotic effects of recombinant peptides, we used the OC as indicator of changes in the osmoregulatory performance of shrimp upon salinity changes. The OC has typically been applied to evaluate the effects of stressors on the physiology of crustaceans (Lignot et al. 2000), but it has also been used to analyze the effects of hormones on

Discussion CHHs are recognized as multifunctional hormones regulating diverse physiological processes in crustaceans (Chung et al. 2010; Webster et al. 2012). Even though previous studies have suggested that CHH and CHH-L isoforms are involved in metabolism, hydromineral balance and stress response in L. vannamei (Lago-Lestón et al. 2007; Liu et al. 2014; Camacho-Jiménez et al. 2015, 2017a, b; Wang et al.

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Fig. 6  Time-course effects of rCHH-B1 and rCHH-B2 on carbonic anhydrase (CA) (a) and N ­ a+-K+-ATPase (NKA) (b) gene expression in posterior gills of shrimp exposed to 8, 26 and 45‰ salinity. ND, no data. Data are expressed as median ± 95% CI (n = 9). Aster-

isks indicate significant differences (p < 0.05) compared to the preinjection group (0  h). Different letters indicate statistical differences (p < 0.05) between groups per sampling time (h)

osmoregulation (Charmantier-Daures et al. 1994; CamachoJiménez et al. 2017a, b). The OC takes into account the contribution of all dissolved osmolytes in hemolymph to the OP, ­ l− being the of which ions account for ~ 90%, with N ­ a+ and C most abundant (Lignot and Charmantier 2015). However, the concentration of each ion in the hemolymph is influenced by its abundance in the environment, related to the salinity and the salt composition of the water (Tantulo and Fotedar 2007; Mena-Herrera et al. 2011). Thus, parallel to the OC, we have determined the ionoregulatory capacity (IC) for N ­ a+ and − ­Cl , for a more direct examination of the effects of rCHHs on the control of hemolymph ion concentrations under different osmotic conditions.

The rCHHs did not modify the OC and ICs in shrimp acutely exposed to 8‰, and all the animals died within 3 h after the abrupt change in salinity. In these conditions, the IC levels for ­Na+ in all exposed shrimp were close to 0, in contrast to C ­ l−, which was hyper-regulated. This behavior was probably due to a reduced ability of L. vannamei to rapidly adjust N ­ a+ and C ­ l− concentrations in hemolymph after the acute exposure to low salinity, as has been observed to occur at salinities below 20‰ (Mena-Herrera et  al. 2011). Under these conditions, the active uptake of salts is increased to cope with the passive loss to the environment, requiring an increase in the CA and NKA activities in osmoregulatory organs (Henry et al. 2012). These increases

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are often accompanied with a rise in ­NH4+ excretion due to protein catabolism, which supplies the energy necessary for the active ion transport and the exchange of N ­ H4+ by + ­Na through the NKA system (Rosas et al. 1999; Díaz et al. 2001). Although shrimp did not show significant changes in CA or NKA expression with the acute change to low salinity, modifications at the activity level cannot be discarded, since a remarkable reduction of hemolymph total proteins (that were not Hc) was observed, probably due to their catabolism. It is possible that the energy supplied by the reserves, including the mobilized glucose at 1 h post-injection, was not enough for the shrimp to be able to cope rapidly with the acute exposure to hypo-osmotic stress, as suggested by the high mortality observed. The mortality could also be explained by the diminished hyper-osmoregulatory capability of large juvenile to sub-adult shrimp in comparison to smaller shrimp that could be observed when cultured in low salinity water, which limits their ability to maintain the internal homeostasis and eventually leads to molt associated mortality (Gong et al. 2004). In fact, even though a great production can be obtained from low salinity (< 5‰) farming of L. vannamei, a reduction of growth and survival have been found under these conditions (Ponce-Palafox et al. 1997; Rosas et al. 2001). The rCHH-B1 and rCHH-B2 peptides promoted transient changes in the osmo-ionic regulation of shrimp under isoand hyper-osmotic conditions, by modifying the IC for ions and the OC of L. vannamei in a differential way depending on the salinity conditions. The osmoregulatory role of CHHs has mainly been studied during the transference of crustaceans to dilute media in eyestalk-ablated animals, showing the ability of these hormones to modify hemolymph OP and ion concentrations, and to partially reverse the osmo-ionic effects of eyestalk ablation (Charmantier-Daures et al. 1994; Serrano et al. 2003). However, the specific mechanisms responsible for those effects remain unclear. Hypothetically, the CHHs could act throughout their hyperglycemic effect by favoring the availability of energy for active osmoregulation, or may directly stimulate the process at a cellular level in the osmoregulatory organs (Spanings-Pierrot et al. 2000). Both rCHH-B1 and rCHH-B2 increased the glucose levels in hemolymph under all experimental salinities, confirming that the stimulation of glucose mobilization from reserves in the tissues to the hemolymph is one of the principal functions of these isoforms, as has previously been detected in L. vannamei acclimated to hyper-osmotic salinity (35‰) (Camacho-Jiménez et al. 2015, 2017b; Wang et al. 2017). However, in posterior gills of the euryhaline crab Pachygrapsus marmoratus, the incubation of tissue with purified CHH, increased the trans-epithelial potential and ­Na+ influx, suggesting a role in the modulation of branchial ion transport (Spanings-Pierrot et al. 2000).

Specific binding sites for CHH were located in gills of C. maenas and C. sapidus, where peptides have the ability to raise cGMP as second messenger (Chung and Webster 2006; Katayama and Chung 2009). In C. maenas, the exposure to low salinity increased the circulating CHH, cGMP and glucose levels in gills, suggesting that CHH acts through branchial membrane-bound guanylyl cyclase receptors (Chung and Webster 2006). Thus, in addition to glycogen storage tissues (i.e., muscle, hepatopancreas), receptors for CHH-B1 and CHH-B2 may be located in the gills of L. vannamei, where they could activate signal transduction pathways using cyclic nucleotides as second messengers to control the cellular ion transport mechanisms. Interestingly, the expression of the ion transport peptide of L. vannamei (LvITP) has been detected in gills at the transcript level, and the silencing of this gene caused branchial hemorrhage (Tiu et al. 2007). Since CHH-B1 has 95.8% identity to LvITP, it is possible that this isoform could act as either an endocrine or paracrine signal in gills. The role of CHH on ion transport has been proposed specifically in the modulation of NKA activity in gills (Spanings-Pierrot et al. 2000; Serrano et al. 2003). Experiments in L. vannamei using a rCHH peptide (rLvCHH) have shown its capacity to increase the NKA activity in shrimp acclimated to 31‰ (Liu et al. 2014). According to our results, NKA expression was clearly up-regulated at 26‰ salinity, the isoosmotic point of L. vannamei, and even more so at 45‰, a stressful salinity that demands osmotic work to balance water and osmolytes in hemolymph and tissues of shrimp (Valdez et al. 2008). These results provide evidence for a potential role of CHH-B1 and CHH-B2 on NKA activity by modulating the α-subunit gene expression. In addition, the CA transcripts were also up-regulated at 45‰ salinity. The neuroendocrine control of CA activity was already proposed by experiments in crabs, in which eyestalk ablation increased the expression and activity of the enzyme, suggesting the existence of an inhibitory neuropeptide in the eyestalks (Henry and Borst 2006; Henry and Campoverde 2006). In contrast, our results suggest that CHH-B1 and CHH-B2 could stimulate CA expression in L. vannamei after acute exposure to hyper-osmotic salinity conditions. The changes in CA and NKA expression could explain the changes in the IC levels for ­Na+ experienced by the shrimp during the acute exposure to high salinity. Since H ­ + and H ­ CO3− may serve as counter + + ions used by apical ­Na /H and ­Cl−/HCO3− exchangers, CA activity can complement the role of NKA in NaCl absorption across the gills (Henry et al. 2012). It would be interesting to determine whether the changes observed in CA and NKA expression are reflected in the enzymatic activities under the same experimental conditions. The changes in CA expression could also be due to alterations in the acid–base status of hemolymph by the exposure to fluctuations in salinity (Henry et al. 2012). Alkalosis has

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been reported in euryhaline crustaceans during low salinity exposure (Henry and Cameron 1982; Whiteley et al. 2001), whereas acidosis has been observed under hyper-osmotic stress (Truchot 1981, 1992; Henry and Cameron 1982). Such disturbances in acid–base homeostasis are, in part, compensated for the exchange of acid/base equivalents with the environment, mediated by CA activity and ion exchange mechanisms at gills (Henry and Wheatly 1992). Acid–base regulation and osmoregulation are necessary processes in the physiology of the crustaceans that are inherently related (Henry and Wheatly 1992). Those mechanisms could be regulated by CHH, since other stressors that affect acid–base equilibrium, such as temperature, hypoxia, and air exposure trigger its secretion (Chang et al. 1998; Chung and Zmora 2008; Shinji et al. 2012). It has been generalized that CA and NKA expression and activity increase in euryhaline crustaceans during acclimation to a low salinity (Henry et al. 2012); however, it seems that in some euryhaline species, both enzymes are induced in response to hyper-osmotic stress (Luquet et  al. 2005; Jayasundara et al. 2007; Roy et al. 2007; Li et al. 2015; Liu et al. 2015). Analyses from metadata suggest that the characteristics following the salinity transfer influence the changes in expression of ion transporter genes of euryhaline crustaceans. Nonetheless, most of the studies have been conducted under chronic, not acute exposure to salinity (Havird et al. 2013). In the present work, we suggest that both NKA and CA activities could be regulated at the transcriptional level in L. vannamei by CHH-B1 and CHH-B2, mainly under hyper-osmotic stress. However, we do not discount the presence of other endocrine molecules in the eyestalk that may be important osmoregulatory effectors, such as the biogenic amine-like neurotransmitters (Lignot and Charmantier 2015). Dopamine (DA) and serotonin have been observed to induce the NKA in gills of L. vannamei (Liu et al. 2009), and receptors for DA acting via cAMP have been located in posterior gills of E. sinensis (Mo et al. 2002). Recently, injection of synthetic red pigmentconcentrating hormone (RPCH), a neuropeptide expressed in XO­–SG, has shown to increase NKA activity in gills of P. monodon shrimp (Sathapondecha et al. 2014). Thus, these endocrine factors could act either in a synergistic or antagonistic manner to CHHs in osmoregulation. Other endocrine structures, such as pericardial organs, could also be involved in this process (Cooke and Sullivan 1982). The changes in the metabolic variables promoted by rCHHs under iso-osmotic and hyper-osmotic conditions could also be an important indication of the osmoregulatory response of L. vannamei, since it could represent an increase in energy expenditure. Besides hyperglycemia, the observed increase in total proteins promoted by the rCHHs at 26‰ salinity and their progressive decrease at 45‰ salinity suggests that CHH-B1 and CHH-B2 not only participate in carbohydrate but also in protein metabolism. These results

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are in agreement with Valdez et al. (2008) who proposed that carbohydrates are the primary source of energy under optimum salinity, whereas protein and lipids are catabolized under high salinity conditions. The role of CHH on lipid metabolism was proposed by Santos et al. (1997), showing that the hormone promotes lipid mobilization from tissues to hemolymph. Also, experiments performed by our group have shown that both rCHH-B1 and rCHH-B2 increased triglycerides in the hemolymph of shrimp under hyper-osmotic salinity (Camacho-Jiménez et al. 2017b; Camacho-Jiménez and Ponce-Rivas, unpublished work). In addition to glucose and lipids, our result suggests that CHH not only stimulates the mobilization of proteins, but also their catabolism under stress conditions. Although the energy obtained from proteins may be used for osmotic work, the increase and decrease of hemolymph proteins could also be associated with tissue volume regulation. Augusto et al. (2009) suggested the possibility of tissue volume regulation through the adjustment of intracellular osmotic effectors, including the small peptides and free amino acid (FAA) pools, whose titers in the hemolymph appear to respond to molecules synthesized by the eyestalks and other neuroendocrine organs (Freire et al. 1995). The measuring of FAA in hemolymph and tissues, as well N ­ H4 + excretion could help to clarify the role of CHHs in protein metabolism at different salinities. In conclusion, CHH-B1 and CHH-B2 are multifunctional peptides that are most likely involved in the control of osmotic and ionic mechanisms in L. vannamei during short-term exposure to salinity stress. The individual effects of these two neuropeptides seem to differ from each other, and seem to be influenced by salinity conditions. These differences in activity may, in part, derive from differences in the sequence and structure of the two neuropeptides (Camacho-Jiménez et al. 2017b). However, it will be necessary to study the hormone–receptor interactions and signal transduction pathways in effector organs in response to diverse experimental conditions, for a better understanding of the biological significance of the CHH/CHH-L neuropeptides in the environmental adaptation of L. vannamei. Acknowledgements  This work was funded by the National Council of Science and Technology of Mexico (CONACyT) (Grant CB2009133958-Z to Ponce-Rivas). The financial support for the doctorate studies of L. Camacho-Jiménez was also granted by CONACyT. We appreciate Dr. John van der Meer for his valuable suggestions in polishing the English in the manuscript. We thank to Ariana Montiel and Gloria Salinas for their help during the sampling procedures.

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