Does the heterotrophic system influence the cellular immune response of Litopenaeus vannamei shrimp? In vitro phagocytosis indices and superoxide anion production comparisons

Highlights

• •Shrimps from clear water system have higher haemocytes values after 60 days.
• •Shrimps have higher haemocyte values after 120 days in heterotrophic system.
• •Shrimps from heterotrophic system have higher phagocytic capacity after 120 days.
• •After 180 days no difference is seen in cellular immune responses between systems.
• •Superoxide anion levels are not affected by any of the rearing systems.

Abstract

Aquaculture production has increased in the last decades, with crustacean production contributing with 9.8% of the total production. However, fisheries and aquaculture sectors present several challenges, such as fish stocks fished beyond biological sustainability, animal diseases, biosecurity, and environmental impact. It is important to improve shrimp production with healthy animals, avoiding environmental impacts, e.g. with the use of heterotrophic rearing system. It is known that the heterotrophic system can stimulate the activation of immune genes, but how it affects the shrimp immune system is unknown. To assess if a heterotrophic system influences the cellular immune response in shrimp, Litopenaeus vannamei shrimp were reared in heterotrophic and clear water systems. Cellular immune response parameters such as total and differential hemocyte counts, phagocytosis indices and the production of the superoxide anion were evaluated after 60, 120 and 180 days. After 60 days, total haemocyte counts were higher in shrimps reared in the clear water system, while after 120 days it was higher in shrimps reared in the heterotrophic system. No significant difference was observed after 180 days. Hyaline, granular and semi-granular cells showed similar behavior, peaking after 120 days in the heterotrophic system. By the 60th day, phagocytic capacity was higher in the heterotrophic system, while no differences were found for the 120th and 180th day. No differences were detected concerning the phagocytic index or superoxide anion production. The heterotrophic system can affect total and differential shrimp haemocyte counts and phagocytic capacity, depending on the period of time they were maintained in this system. However, the phagocytic index and superoxide anion production are not affected by the heterotrophic system at the time points evaluated herein.

1. Introduction

In 2016, aquaculture activities produced a total 80 million of tonnes of aquatic organisms, totaling USD 231 billion, with crustacean farming accounting for 9.8% and 24.6% of this production in volume and value, respectively [16]. Besides comprising an important source of protein, crustaceans are an important revenue in developing countries for people engaged in the value chain. The crustacean sector generates high value products which enable producers to buy lower value products in the world market, contributing to food security and occupation in both producing and exporting countries [7]. Some of the major issues that constrain aquaculture expansion are transboundary aquatic animal diseases, biosecurity concerning shrimp production in intensive systems [7] and environmental impacts caused by the discharge of nutrient-rich waste products into water bodies [13]. Zero-exchange aquaculture systems with no or minimal water exchange during the rearing cycle represent a solution to these problems [2,18]. These systems combine the removal of water nutrients and the production of microbial biomass, known as bioflocs or heterotrophic systems, which can be used in situ by cultured species as additional nutrient sources [13,18]. This, in turn promotes biosecurity through pathogen exclusion [13] and provides immunostimulatory compounds, improving shrimp health [12].

Improvements in shrimp health are linked to the environmental and nutritional modulation on the immune system, thus controlling and preventing diseases [5]. It can be stimulated with a wide variety of agents, such as the use of putative vaccines [31], in addition of probiotic sources - microalgae, yeasts, bacteria – in rearing systems [51] or even with plant extracts [37,47,48,50] and, usually with commercial immunostimulants in shrimp chows [29].

Since invertebrates lack a true adaptive immune response system, they rely on the innate immune system against pathogen invasion [46]. This response consists of phagocytosis, encapsulation, nodule formation, clotting, agglutination and microbicidal activity [17,35,46] displayed by the hemolymph and haemocytes [1,28]. Three main haemocyte populations are usually identified in crustaceans, hyaline or agranular, semi-granular (with small granules) and granular (with large granules) cells [4,27,39].

The purpose of the present study was to identify the effects of different culture systems on the innate immune response of Litopenaeus vannamei shrimp. To this end, we examined total haemocyte counts (THC) and differential haemocyte counts (DHC), phagocytosis indices and superoxide anion production as immunological indicators of the health status of this penaeid shrimp [9,30,36,41].

2. Material and methods

2.1. Shrimp source and experimental conditions

Litopenaeus vannamei shrimps from a commercial hatchery (Aquatec Industrial Pecuária Ltda., Barra do Cunhaú, RN, Brazil) were stocked in triplicate tanks (500 L) in each system at a density of 150 kg m⁻³ for 180 days. This study was performed in a recirculated aquaculture system equipped with a bubble bead filter (BBFXS 8000, Aquaculture Systems Technologies, LLC. New Orleans, LA, USA) to clear water system. Air stones supplied oxygen for clear water and heterotrofhic systems.

Shrimps were fed a commercial feed ad libitum (Potimar 38 Active - Guabi®, Campinas, SP, Brazil) eight times a day. Molasses was added to the heterotrophic system at a rate of 50% of the feed weight as a supplemental carbon source to the heterotrophs [14].

Water temperature (°C), pH, salinity (ppt), dissolved oxygen (mg L⁻¹) and settleable solids (mL L⁻¹) levels were monitored daily. Ammonia-N (mg L⁻¹) was monitored daily during the first ten weeks and weekly . Concerning the heterotrophic system, 5% of the water volume was exchanged at the end of the cycle to avoid excessive nitrite-N levels.

Our aim was to demonstrate how the immune system would behave during commercial farming. Since the shrimps take six months to reach their selling weight, we decided to separate this period into three study periods, in order to evaluate when shrimps are more susceptible to infection.

2.2. Haemolymph collection and cell counting

A total of 20 individuals from each tank of both systems were sampled on the 60th, 120th and 180th days, weighed and anaesthetized in chilled sea water for 30 s [31]. Haemolymph was withdrawn from the ventral sinus of each shrimp in 1 mL sterile syringes (25 gauge needle) containing an anticoagulation solution (50 mmol L⁻¹ NaCl, 10 mmol L⁻¹ KCl and 10 mmol L⁻¹ EDTA at pH 7.3) [40].

The hemolymph samples were randomly allocated to two groups, where 10 samples were used in the phagocytosis assay and 10 in the superoxide anion production assay. A portion of the haemolymph of all individuals was analyzed for total and differential haemocyte counting.

For the cell counts, a drop of haemolymph was placed on a hemocytometer and observed under a phase-contrast microscope (Standard 25 ICS, Carl Zeiss™, Germany) [6]. Hemocytes were differentiated according to Bachère et al. [4]; Martin and Graves [27] and van de Braak et al. [39], where hyaline cells have no citoplasmatic granules, semi-granular cells contain small cytoplasmatic granules and granular cells display large cytoplasmatic granules.

2.3. Phagocytosis assay

A modified phagocytosis assay was employed [19,21,34], in which 100 µL of haemolymph were placed in a round cover slip on a 24-well culture plate and incubated in a wet chamber for 1 hour for cell adherence and spreading. Subsequently, Saccharomyces cerevisiae at a ratio of 10 yeasts for one phagocyte were added. The plate was then incubated in a wet chamber for 3 h, followed by cover slip fixing with paraformaldehyde (4%) and rinsing in distilled water. The cover slips were rinsed in May-Grünwald-Giemsa stainand mounted on a microscope slides. The slides were then analyzed under a Standard 25 ICS microscope (Carl Zeiss™, Germany), where a total of 100 haemocytes were counted to calculate the phagocytosis indices, as follows:

$$\begin{array}{ccc}\hfill \text{Phagocytic}\text{Capacity}\left(\text{PC}\right)& =& (\text{No}.\text{of}\text{phagocytic}\text{haemocytes}\times 100)\hfill \\ & & \times {(\text{no}.\text{of}\text{total}\text{haemocytes})}^{-1}\hfill \end{array}$$

$$\begin{array}{ccc}\hfill \text{Phagocytic}\text{Index}\left(\text{PI}\right)& =& (\text{total}\text{number}\text{of}\text{phagocitosed}\text{yeast})\hfill \\ & & \times {(\text{no}.\text{of}\text{phagocytosing}\text{haemocytes})}^{-1}\hfill \end{array}$$

2.4. Superoxide anion production (Nitroblue tetrazolium –NBT assay)

Superoxide anion production was assessed after the reduction of nitroblue tetrazolium (NBT) to insoluble blue Formazan. To this end, 100 µL of haemolymph were transferred to a 96-well microtiter plate, in triplicate, for each animal plus 100 µL of Van Harrevald´solution (VHS, 0.2 mol L⁻¹ NaCl, 0.1 mol L⁻¹CaCl₂, 2 mmol L⁻¹MgCl₂, 5 mmol L⁻¹ KCl, 2 mmol L⁻¹ NaHCO₃, pH 7.4) for 30 min at room temperature to form a cell layer. The supernatants were then removed and replaced with 100 µL of an NBT solution (0.1% of NBT and 0.2% de Saccharomyces cerevisiae) for 2 h at 37 °C. Each well was then washed with 70% methanol and absolute methanol. Finally, 120 µL of 2 mol L⁻¹ KOH followed by 140 µL of dimethyl sulfoxide (DMSO, SIGMA, USA) were added to dissolve cytoplasmic formazan and absorbances were determined at 63 0 nm using a microplate reader (Sunrise, Tecan, Austria) [21].

2.5. Statistical analyses

An ANOVA and nonparametric Kruskal-Wallis tests were applied to identify the effects of the shrimp rearing systems on their performance and immune parameters, respectively. The Statistica 6.0 software (StatSoft, Inc. Tulsa, OK, USA) was used for all statistical analyses. Results are presented as means and standard deviation (sd) for parametric analyses and medians and maximum and minimum values for nonparametric analyses. Differences were significant at p < 0.05.

3. Results

3.1. Water quality and shrimp performance

The water quality parameters (mean ± sd) in the recirculating and the heterotrophic systems were determined as follows: temperature: 27.8 ±1,1 and 27.9 ± 1.4 °C, dissolved oxygen: 4.7 ± 0.7 and 4.6 ± 0.7 mg L⁻¹; pH: 7.8 ± 0,4 and 7.8 ± 0.4, salinity: 32.9 ± 1.6 and 33.8 ± 2.2 ppt, ammonia-N: < 0.5 and 1.0 ± 0.7 mg L⁻¹, settleable solids: 26.2 ± 28.1 mg L⁻¹.

Shrimp performance (Table 1) assessments revealed that the growth rates of the shrimps in the heterotrophic system were higher after 120 days of culture (p = 0.000) compared to the growth rate of the shrimps reared in the clear water system (13.29 ± 3.07 and 9.90 ± 3.00 gm, respectively). However, after 180 days of culture the mean weight of the shrimp in the clear water system was higher (p = 0.001) than those reared in the heterotrophic systems (18.68 ± 4.02 and 16.29 ± 3.45 g, respectively).

Table 1.

Weights (mean ± sd) of the juveniles of the shrimp Litopenaeus vannamei reared in clear water and heterotrophic systems at day 60th, 120th and 180th.

	Day 60	Day 120	Day 180
Clear Water	4,35 ± 1,11 Aa	9,90 ± 3,00 Bb	18,68 ± 4,02 Cd
Heterotrophic	5,54 ± 1,28 Da	13,29 ± 3,07 Ec	16,29 ± 3,45 Ed

Weights from shrimp reared at the different systems, at the different sampling times. Different capital letter means p < 0.05 among kinetics, and different small letter means p < 0.05 between treatments.

3.2. Cell counting

Only significant differences are reported. The THC at the clear water system decreased from 30.87 × 10⁴ cells mL⁻¹ (range 3–386 × 10⁴ cells mL⁻¹) on the 60th day to 14.0 × 10⁴ cells mL⁻¹ (range 0.75–58.75 × 10⁴ cells mL⁻¹) on the 120th day, followed by an increase to 225.5 × 10⁴ cells mL⁻¹ (range 111.0–1020.0 × 10⁴ cells mL⁻¹) on the 180th day. The THC in the heterotrophic system increased from the 60th day (14.0 × 10⁴ cells mL⁻¹, range 3–34 × 10⁴ cells mL⁻¹) to the 120th day (111.0 × 10⁴ cells mL⁻¹, range 35–189.5 × 10⁴ cells mL⁻¹), increasing further on the 180th day (189.5 × 10⁴ cells mL⁻¹, range 81–708 cells mL⁻¹). On the 60th day, the THC were higher in the clear water system, while on the 120th day higher THC were observed in the heterotrophic system (Fig 1).

Fig. 1.

Total Hemocyte count: Total hemocyte count (n°10⁴ cells ml⁻¹) from Litopenaeus vannamei shrimps reared in clear water and heterotrophic systems after 60, 120 and 180 days. Shrimps reared in heterotrophic system showed higher mean values in all time points evaluated, but with high standard deviations.

The number of hyaline cells (HC) in the clear water system increased after 180 days, while the number of granular cells (GC) increased from the 60th to the 120th day, decreasing on the180th day. The number of semi-granular cells (SGC) decreased over time in both systems, although no significant difference was observed in the heterotrophic system from the 120th to the 180th day. The HC in the heterotrophic system increased from 60th to 120th day. GC decreased from the 120th to the 180th day. When comparing both systems, HC were the same on the 60th and 180th days, and higher in the heterotrophic system on 120th day . GC were also the same on the 60th and 180th days, but higher in the clear water system on the 120th day, similarly to the SGC (Table 2).

Table 2.

Differential hemocyte count (%) of the shrimps reared at the different systems.

		60 Days	120 Days	180 Days
Clear water system	Hyaline Cells (HC)	61.13 (Range 50.0–94.82) Aa	58.54 (Range 25.58–92.28) Ab	80,93 (Range 65.33–91.53) Bd
	Granular Cells (GC)	22.27 (Range 1.95–33.33) Ce	25.0 (Range 5.51–66.67) Df	13,59 (Range 7.33–28.0) Eh
	Semi-granular Cells (SGC)	16.6 (Range 0–28.57) Fi	8.33 (Range 0–20.45) Gj	4.10 (Range 0.52–10.67) Hl
Heterotrophic system	Hyaline Cells (HC)	71.43 (Range 50.0–85.71) Ia	76,56 (Range 62.86–89.85) Jc	82.51 (Range 68.31–91–84) Jd
	Granular Cells (GC)	14.28 (Range 0–28.57) KLe	19.05 (Range 7.25–34.28) Kg	14.9 (Range 6.8–28.41) Lh
	Semi-granular Cells (SGC)	14,28 (Range (4,76–33.33) Mi	2.9 (Range 1.64–10.14) Nk	2,81 (Range 0.89–10.81) Nl

Differential hemocyte count (percentage) from shrimp reared at the different systems, at the different sampling times. Different capital letter means p < 0.05 among kinetics, and different small letter means p < 0.001 between treatments.

3.3. Phagocytic assay

Only significant differences detected in the phagocytic assay are reported. In the clear water system, phagocytic capacity (PC) increased from the 60th to the 120th day, subsequently decreasing, while no changes between the 60th and the 120th day were noted for the heterotrophic system, decreasing on the 180th day. The comparison between both systems indicates that the PC at the heterotrophic system was higher on the 60th day compared to the clear water system (Fig 2A).

Fig. 2.

Phagocytic capacities (%) and phagocytic indices of haemocytes of Litopenaeus vannamei reared in clear water and heterotrophic systems and analyzed after 60, 120 and 180 days. Data collected after three hours of phagocytosis stimulation with Saccharomyces cerevisiae yeasts: A- In all time points evaluated there is no difference phagocytic capacities (%) between the rearing systems. B- Phagocytic indices have no difference among time points evaluated excepting for haemocytes of shrimps reared in heterotrophic system during the first 60 days (p < 0.05).

The phagocytic index (PI) of the clear water system decreased from the 60th to the 120th day while, in the heterotrophic system, PI decreased from the 60th to the 120th day, further decreasing from the 120th to the 180th day. No differences between the two systems were detected (Fig 2B).

3.4. Superoxide anion production

In this assay, the higher the absorbance, the higher the production of the superoxide anion. In the clear water system, superoxide anion production decreased from the 60th to the 120th day and increased from the 120th to the 180th day, reaching the same absorbance as on the 60th day. In the heterotrophic system, superoxide anion production decreases from the 60th to the 120th day. No differences between the two systems were detected (Fig 3).

Fig. 3.

Superoxide anion production by Litopenaeus vannamei haemocytes reared in clear water and heterotrophic systems. Analyses at each time point – 60, 120 and 180 days of shrimp production: Superoxide anion production at the different sampling times. There is no differences between the two systems.

4. Discussion

Water quality was maintained within the recommended range for the Litopenaeus vannamei, except for settleable solids, which were recorded at levels as high as 150 mL L⁻¹ at the end of the rearing trial, above the recommended target of 10 ml L⁻¹ [33]. High levels of suspended solids may cause gill clogging and compromise shrimp oxygen uptake [32], which may explains the lower weight of shrimp reared in the heterotrophic system.

Concerning the THC, its oscillation in clear water shrimps at different timeframes can be explained as a physiological effect, as THC can vary due to intrinsic or extrinsic factors, such as molt stage and bacterial infection [10,43].

According to Johansson et al. [22], THC and DHC variations are probably regulated by their release from hematopoietic tissue. The THC of animals reared in the heterotrophic system increased over time, implying a production stimulus or THC release [22], although shrimps in clear water system presented higher THC than heterotrophic shrimp after 60 days of rearing. This has also been reported for shrimps exposed to different microorganism [25,48,49]. The lower THC in the heterotrophic system after 60 days may be due to increased bacterial levels in digestive shrimp tracts, leading to hemocyte migration to the mucosa layer, reducing haemolymph values [11]. Further research on hematopoietic stimulation in shrimp reared in heterotrophic systems will help address this issue.

The THC in the heterotrophic system was higher on the 120th day, suggesting a THC production or release stimulus. We believe this may be due to contact with biofloc for a substantial period of time, thus stimulating immune system reactions, such as hematopoiesis [45].

On the 180th day, no significant difference was observed between the THC of both systems, with similar results found for he same specie by different authors. [23,37,42]. This phenomenon may be due to the development of an immunostimulant tolerance, as reported for fish [8], and suggested for shrimp [42]. Comparing THC values between the 60th and 180th days, a 5-fold increase in THC values was observed in the clear water system, increasing to 13.2-fold in the heterotrophic system, implying a hematopoietic stimulus.

DHC data for L. vannamei reported in the literature are conflicting, probably due to assay differences, such as the type of investigated immunostimulant. Furthermore, differences between THC and DHC among control animals have also been reported [3, 29]. Liu et al. [26] suggest that this may be due to environmental factors, population variability or a combination of both. Furthermore, our results indicate high individual variations, was also reported by Li et al. [24], difficulting literature data comparisons.

Phagocytosis is a highly conserved mechanism used by cells to ingest microparticles, such as pathogenic microorganisms and cell debris [44]. PC values increased from the 60th to the 120th day and decreased on the 180th day in both systems. Our hypothesis for increased PC values comprises immune system maturation, while decreases may be due to sexual maturation or immune system aging . In the heterotrophic system, PC decreases suggest immune suppression, as reported for shrimps challenged with WSSV (White spot syndrome virus) [20], or immunostimulant tolerance as reported for fish [8], while some authors suggest the same may occur in shrimps [42] . In addition, no significant differences in PI values were observed when comparing both systems, although a significant decrease in PI values at different sampling times in both systems occurred, suggesting that this decrease may be physiological.

The production of the superoxide anion varied during the trial, suggesting physiological reasons. No significant difference were observed for this parameter between both systems, as reported in different situations, such as shrimps fed biofloc [15], immersed in water contaning a leaf hot-water C. kanekirae extract [47] and fed diets containing B. subtilis E20 [38].

5. Conclusion

The heterotrophic system improved the number of phagocyting hemocytes and phagocytic indices at certain time points, but with no effects on the production of the superoxide anion. Research challenging animals reared in heterotrophic systems with pathogens would be interesting to address whether heterotrophic systems are able to enhance the shrimp immune system.

Funding

This work was supported by Fapesp [Grant number 2010/04293-4].

Credit author statement

Renata Stecca Iunes: experimental design, sampling collection, cell counting, phagocytic assay, anion superoxide assay, writing original draft and final version of the manuscript.

Paola Cristina Branco: sampling collection, cell counting, phagocytic assay, anion superoxide assay, writing review of the manuscript.

Leandro Nogueira Pressinotti: Statistical analysis, writing review of the manuscript.

Rodrigo A. P. de L. F. de Carvalho: experimental design, analysis of water quality and animals’ performance, writing review of the manuscript.

José Roberto Machado Cunha da Silva: teacher advisor, experimental design, writing review of the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.