DOI: 10.31038/AFS.2021354

Introduction

Edwardsiella tarda is one of the most significant bacterial pathogens to fish aquaculture. This Gram-negative, intracellular bacterium is the causal agent of the “edwardsiellosis” which cause generalized septicemia and lesions on internal organs. It has been reported worldwide in economically important fish species, including Japanese eel (Anguilla japonica), red sea bream (Pagrus major), yellowtail (Seriola quinqueradiata), channel catfish (Ictalurus punctatus), turbot (Scophthalmus maximus) and tilapia (Oreochromis sp.) [1,2]. This infection leads to enormous economic losses in cultured seawater and freshwater fish [1].

Different strategies have been attempted to treat and prevent E. tarda infections, including antibiotics and chemicals. However, overuse or abuse of these can result in the selection and development of drug resistant pathogens [3,4]. As a promising alternative, immunostimulants may represent a safe and effective treatment to enhance an antimicrobial response for preventing fish infection by a wide range of pathogens.

Synthetic growth hormone (GH) secretagogues (GHSs) consist of a family of ligands, initially termed GH-releasing peptides (GHRPs) [5] which are recognized by the GHS receptor (GHS-R). GHS-R is expressed predominantly in the brain and pituitary gland, and in immune system cells from mammals and fish, suggesting a relation between ligands for these receptors and the immune system [6]. Growth Hormone-Releasing Peptide 6 (GHRP-6) is a six amino acid synthetic peptide with demonstrated stimulatory effects on innate and adaptive immune system in teleost fish [7,8]. The aim of this study was to assess the antimicrobial effects of GHRP-6 against bacterial infection in tilapia, as well as to measure gene expression of some immune-related genes before and after E. tarda infection.

Materials and Methods

Fish and Peptide Source

Juvenile tilapias (Oreochromis niloticus, male, ~100 g) were obtained from the Aquaculture Research Station at the Center for the Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. Fish were kept alive in aerated freshwater in 120 L tanks under a 12 h light: 12 h dark photoperiod. They were fed commercial dry diet for fish (CENPALAB, Habana, Cuba). Water temperature was maintained at 26-28°C. Fish were acclimated for one week before each experiment. All animal experiments were previously approved by the Ethics Committee of the Center for Genetic Engineering and Biotechnology, Havana, Cuba. The described work has been carried out in accordance with EU Directive 2010/63/EU for animal experiments.

GHRP-6 (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW = 872.44 Da) with a purity > 99% was provided by Sigma-Aldrich, USA.

Fish Treatment with GHRP-6

Fish (26 tilapias per group) were injected by intraperitoneal route (i.p) during 7 days. One group of tilapias received GHRP-6 (0.2 µg/body weight (bwg)) in a total volume of 100 µL of PBS. The Control group was injected with the same volume of PBS. The dose employed for GHRP-6 was chosen based in previous experiments performed in the laboratory [9]. Spleen samples (n=6) were taken at 24 and 48 hours after the last injection. Tissue samples were stored in Ambion RNAlater (AppliedBiosystems, USA) at -20°C until use for RNA isolation.

Also, at 48 hours after the last peptide injection, a challenge was performed using an E. tarda strain by immersion bath as previously described by [10], with minor adjustments. Edwardsiella tarda strain was obtained from glycerol stock cultures stored at -80°C. Briefly, Nile tilapia juveniles (n=13 per group) were exposed to 108 CFU/mL of E. tarda (diluted in sterile PBS) by immersion bath during 1 h in a total volume of 40 L with aeration. After immersion bath, both groups were moved to 250 L aerated freshwater tanks in an isolated water flow system and kept in those conditions until sampling of gills and spleen (8 fish per group). The rest of the animals were daily checked until day 5th day when mortality started to occur. Two additional tanks with fish (n=7 per group: PBS and GHRP-6) were used as non-challenge controls of mortality. After the challenge concluded, waste water was treated with chlorine to avoid the releasing of live bacteria.

Spleen was sampled at 24 and 48 hours after challenge (hac) for gene expression analysis, as described above. To evaluate the antimicrobial effects of GHRP-6 on gills, samples were taken at 48 hours after the challenge (hac), under aseptic conditions and washed in 1 mL of 0.9% NaCl. Afterwards, this wash was serially diluted in sterile PBS, plated in tryptic soy agar (TSA) (Merck) and grown overnight at 37°C. The identity of the colonies grown in specific culture medium was confirmed by an API20E test (Biomérieux) and Gram staining.

Gene Expression Analysis

Tissue samples were processed on a Tissue Lyser unit (Qiagen, Hilden, Germany), extracting total RNA with RNeasy® Plus (QIAGEN GmbH, Germany) using the Quiacube platform. RNA purity and yield were determined using a NanoDrop Spectro-photometer (NanoDrop Technologies, USA). First-strand complementary DNA (cDNA) was synthesized from 1 μg total RNA using Superscript III First-Strand Synthesis Supermix for RT-PCR (Invitrogen Technologies, Carlsbad, California, USA) following fabricant ́s instructions.

Transcripts levels in the sampled organs were assessed by qPCR analysis. Gene’s sequences were obtained from the US National Center for Biotechnology Information database (NCBI). Specific primers were designed, using the web application Primer3 [11]. Each experimental sample was run in triplicate on 96-well plates and qPCR reactions were completed on the LightCycler® 480 II (Roche). All PCR reactions were of 20 µL volume and contained: 10 µL of LightCycler® 480 SYBR Green I Master 2x (Roche, Germany), 300 nM each primer (10 pmol/µL) and 4 µL of cDNA (50 ng/µL diluted 1:25 in RNase free water). For each plate, triplicate wells of a calibrator and RNA only control were also present. The program used for all qPCR reactions was as follows: pre-incubation at 95°C for 5 min followed by 50 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 s and extension at 72°C for 30 s. A melting curve was completed for every run from 72 to 95°C with a read every 5 s. Product specificity was determined through single PCR melting peaks. All qPCR data was analyzed using REST 2009 v2.0.13 [12]. Differences were expressed as fold changes. Gene expression was normalized to the references genes EF-1α and β-actin.

Statistical Analysis

The effects of GHRP-6 on the number of CFU in gills, was assessed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA). The normal distribution of data was analyzed with D’Agostino Pearson’s test and the variance homogeneity with Bartlett’s test. A Student’s t parametric test was performed as data followed normal distribution for comparisons in each time point. For qPCR analysis, reactions efficiency was calculated using LinReg software and data were analyzed using REST 2009 software v2.0.13 [12,13]. This software calculates the relative expression to a control condition after normalize with the efficiency of the selected references genes. Differences were expressed as fold changes relative to Control group. A significance level of 0.05 was used for all analyses.

Results

After the last peptide injection with GHRP-6, the transcripts levels of IL-1β relative to Control group were up-regulated in spleen both at 24 (2.37 ± 0.58) and 48 (2.27 ± 1.64) hours. Also, Oreochromicin III was significantly up-regulated at 48 hours after treatment (5.99 ± 3.48). However, no significant differences were observed for Oreochromicins I and II and Granzyme transcripts (Figure 1).

Figure 1: Gene expression analysis in spleen of tilapia (Oreochromis sp.) after treatment with GHRP-6 during seven consecutive days by i.p injection. Samples were taken at 24 and 48 hours after the last peptide injection. Fold change (FC) with respect to Control group were calculated by REST 2009 (FC>1 up-regulated; FC<1 down-regulated) and were expressed as mean ± SD (n=6). The asterisks indicate statistically significant differences relative to Control group, *(p < 0.05).

Once confirmed the positive modulation of a pro-inflammatory cytokine and an antimicrobial peptide in spleen, we evaluated the antimicrobial effect promoted by GHRP-6 after a challenge trial with E. tarda. With this aim, fish were challenged 48 hours after the peptide treatment with an E. tarda strain. Spleen samples were collected at 24 and 48 hours after challenge, while gills washes and were taken at 48 hours.

GHRP-6 treatment exerts a differential modulation over Oreochromicins, Granzyme and IL-1β mRNA expression profiles in spleen after E. tarda infection. Oreochromicin I was up-regulated (7.49 ± 3.32) at 48 hours, while, Oreochromicins II and III were down-regulated at 24 hours (0.12 ± 0.17 and 0.09 ± 0.07, respectively). On the other hand, IL-1β was statistically down-regulated (0.067 ± 0.002) at 48 hours (Figure 2).

Figure 2: Gene expression analysis in spleen of tilapia (Oreochromis sp.) after treatment with GHRP-6 (during 7 consecutive days by i.p injection) and challenge with an Edwardsiella tarda strain. Twenty-four and 48 hours after the last peptide injection, fish from Control and GHRP-6 treated groups (13 fish per group) were exposed for one hour to 1 × 10⁸ CFU/mL of E. tarda by immersion bath. Gene transcription was analyzed by qPCR in spleen sampled at 24 and 48 h after challenge. Fold change (FC) with respect to Control group were calculated by REST 2009 (FC>1 up-regulated; FC<1 down-regulated) and were expressed as mean ± SD (n=8). The asterisks indicate statistically significant differences relative to Control group, *(p < 0.05), **(p < 0.01) and ***(p < 0.001).

In gills washes, there were statistically significant lower CFU/mL of bacteria in group treated with GHRP-6 (6.8 x 104 ± 8.6 x 103 CFU/mL) compared to the Control group (3.9 x 105 ± 6.4 x 104 CFU/mL) (Figure 3). We observed clinical signs of the disease in some animals of the Control group such as loss of pigmentation and petechial hemorrhage in fin and skin, aspects reviewed by [1] as evidence of the infection. We started to observed mortality in Control group of infected fish since the 5th day, being 4/7 in Control group and 0/7 in GHRP-6 treated group.

Figure 3: Gills’ bacterial load in Control and GHRP-6 treated groups after treatment with GHRP-6 (during 7 consecutive days by i.p injection) and challenge with an Edwardsiella tarda strain. Forty-eight hours after the last peptide injection, fish from Control and GHRP-6 treated groups (13 fish per group) were exposed for one hour to 1 × 10⁸ CFU/mL of E. tarda by immersion bath. Total number of CFU/mL in gills washes were counted at 48 h after challenge. Data represent the mean ± SD (n=8) of three independent experiments. The analysis of data was performed using a Student’s t test. The asterisks represent statistically significant differences between groups, *(p < 0.05).

Discussion

Edwardsiella tarda is a Gram-negative intracellular pathogen which causes enormous economic losses in cultured seawater and freshwater fish [14]. Infection by E. tarda may produce severe lesions in internal organs such as spleen [15-17]. Gills constitutes one of the entry routes of E. tarda infections in fish [18], thus, the use of immunostimulants with antimicrobial effects for preventing infestations could be promising.

Previously, it has been demonstrated that GHRP-6 is a growth hormone secretagogue that improve different innate and adaptive immune system parameters in aquatic organisms [7,8,19]. More recently, we provided new evidence of a direct link between GHRP-6 treatment and enhanced antimicrobial peptides transcription in tilapia [20]. In this study we shed light over the effect of this peptide as stimulator of the antimicrobial immune response in tilapia with and without the presence of a challenge with E. tarda.

Spleen constitutes one of the major lymphoid tissues in fish and also is affected by the infection with bacterial pathogens, such as E. tarda [21]. GHRP-6 statistically up-regulated the mRNA levels of IL-1β in tilapia spleen. Our results are in agreement with previous studies where IL-1β was up-regulated after the administration of immunostimulants [22,23].This cytokine is a critical mediator of the inflammatory response against microbial invasion and can stimulate the immune response by activating lymphocytes or by inducing the release of other cytokines capable of triggering macrophages, NK cells and lymphocytes (Yuan et al., 2008; [18,24]. Also, Oreochromicin III was up-regulated after the treatment with the GHS. Previous studies have demonstrated that this antimicrobial peptide has immunomodulatory effect in mammals. Oreoch III stimulated the release of IFN-γ from mice splenocytes [25].

In the current study, Oreochromicins I, II and III were differentially regulated by GHRP-6 in spleen after the challenge with E. tarda. Therefore, Oreochromicins could be involved in the elimination of this pathogen in tilapia spleen and other tissues. These antimicrobial peptides belong to the piscidin-like family and are expressed in different cell types such as neutrophils, granular eosinophils, monocytes and macrophages [26]. [27] demonstrated strong antimicrobial activity of Oreochromicin II against E. tarda in vitro. Recent studies revealed that GHRP-6 stimulates the antimicrobial immune response against Pseudomonas aeruginosa in tilapia and up-regulated the transcription of Oreochromicins as well in the presence of this pathogen [20].

Our results revealed that the treatment with GHRP-6 statistically decreased the bacterial load of E. tarda in gills washes 48 hours after the challenge as we observed a significant up-regulation of the mRNA levels of Oreochromicin I in spleen. Fish´s gills possess different cells populations and a mucus layer associated to it, which contains different antimicrobial molecules that contribute to the bacterial clearance [28,29]. The obtained results suggested that GHRP-6 may stimulate the antimicrobial response which limits the growth of the bacteria in gills, and thus could restrict the bacterial infection. Previous reports have shown that AMPs can protect against infections by E. tarda in fish [30]. Further studies will be conducted in order to gain knowledge at the histological level. In addition, after the challenge, we observed a down-regulation of IL-1β. Previous studies have demonstrated that when the expression of this cytokine decreases significantly at 48 hours, it is possible that this has increased within a few hours of completing the challenge. For example, [31], showed that in a challenge with Aeromonas salmonicida by intraperitoneal injection, IL-1β levels in spleen were up-regulated at 2 and 6 hours after the infection, but at 24 hours did not showed significant differences. Moreover, in a challenge carried out with E. tarda by intramuscular injection, an increase in IL-1β expression was obtained at 6 and 24 hours, in animals previously immunized with the flagellar protein FlgD by intramuscular injection [32-34].

The results of this trial suggest that the induction of IL-1β could be transient and that other effector components of innate immunity are participating in the protection of the organism. Various authors showed that when the expression of this cytokine decreases significantly at 48 hours, it is possible that this has increased within a few hours of completing the challenge.

In summary, our results suggested novel roles of GHRP-6 in tilapia as possible immunostimulant, which is able to promote the antimicrobial activity against some Gram-negative intracellular bacterial infections, such as E. tarda. Currently we are planning to evaluate the protective effect of this GHS at the histological level when administered in the presence of pathological bacteria such as E. tarda. Also, we are developing an oral formulation in order to evaluate its immunostimulant effects in long term experiments as part of the diet and its effects on guts bacterial load after a bacterial challenge. These studies will allow us to gain a deeper knowledge about the molecular basis of GHRP-6 mechanism in relation with antibacterial response in fish for its potential application in aquaculture.

Author Contributions

LH performed majority of experiments, contributed to experimental design, wrote the first draft and reviewed the final version of the manuscript. HC, ANR, DP, AM, LB, FH, OR, SP contributed with the development of the experiments. JV contributed with the development of the experiments and reviewed the final version of the manuscript. ME, contributed to experimental design and reviewed the final version of the manuscript. RM contributed to experimental design, wrote the first draft of the manuscript and reviewed the final version of the manuscript.

Data Availability

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

Keywords

GHRP-6, Antimicrobial activity, Edwardsiella tarda, Tilapia

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DOI: 10.31038/AFS.2021353

Abstract

Physico-chemical parameters, mollusc distribution and diversity in Shiroro Lake, Minna, Niger State was investigated between the months of March and August, 2017 spanning through the wet and dry seasons. Water samples for physico-chemical parameters and snails samples were collected and identified on monthly basis following standard methods. From the results; the temperature ranged from 26.5-32°C. The pH was all basic throughout the period of the study which ranged from 8.5-9.2 across all the stations. Dissolved Oxygen concentration ranged from 2.6-5.2 mg/L, while the BOD ranged from 2.2-4.9 mg/L. Nitrate level (0.43-0.83 mg/L) was high, while phosphate concentration (0.24-0.42 mg/L) was relatively low in all the sampling stations. A total number of 7 snail species were encountered. Station 3 had the highest number (403), station 2 with 363 species and station 1 had the lowest with 345 species. The family Viviparidae and Bithyniidae has 2 species each. The Bellamya phthinotropis has the highest number of species followed by Bellamya capillata; and the lowest was recorded among the species of Gebtella barthi. In general, the abundance of snails was higher during the rainy season than during the dry season. Taxa richness determined as Margalef index showed significant difference among the sampling stations. Similarly, diversity indices (Shannon Wiener, Simpson, dominance) also showed significant difference among the sampling stations. Lower values of diversity and Eveness indices were recorded at station 1. Station 2 had the highest record of diversity and eveness indices. Shiroro Lake is moderately organically polluted and adequate measures should be taken to check-mate this.

Keywords

Shiroro Lake, Physico-chemical parameters, Molluscs, Pollution and water quality

Introduction

Seasonal variations, pollution and its effects have over the years influenced the chemical contents of water and the survival of biotas in the aquatic environment. This is why it is difficult to understand the biological phenomenon fully because the chemistry of water levels tells much about the metabolism of the ecosystem and explains the general hydro-biological relationship (Basavaraja et al., 2011). Due to the pressures of increasing population and developing economy all over the world, the present situation of water quality management is far from satisfactory (Sawaya et al., 2003). Organic pollution is rampant in municipal water bodies posing health hazards to the neighbouring communities. Also, faecal pollution of drinking water causes water borne disease which has led to the death of millions of people [1]. The quality of drinking water is a complex issue and vital elements of public health. Poor water quality is responsible for the death of an estimated few million children annually (Holgate, 2000). The interaction of physical and chemical properties of water have a significant role in the composition, distribution and abundance of aquatic organisms which are therefore, used to determine the water quality and structural composition of aquatic community (Youne et al., 2003). Some of the major physico-chemical parameters that indicate water quality are the dissolved oxygen (DO) and biological oxygen demand (BOD). The dissolved oxygen is important in the natural self-purification capacity of the river; and the BOD is often used as a measurement of pollutants in natural and waste waters and to assess the strength of waste, such as sewage and industrial effluent waters [2]. BOD is an important parameter of water indicating the health scenario of freshwater bodies [3].

Gastropods are single-valve, soft-bodied class of animals in the phylum Mollusca. It is the largest, extremely diverse taxa that includes over 40,000 species of which 5,000 are fresh water snails found in wetlands like lakes, ponds and streams worldwide. Freshwater snails are an important food source for many fish, turtles, and other species of wildlife. As a result of their sensitivity to certain chemicals, many species are excellent water-quality indicators. Over the years, conservation and recovery efforts for freshwater snails include artificial culture or heliculture, water pollution control, and most importantly, habitat protection and restoration. Cleaning waterways not only improves the habitat for snails and other aquatic life, but it also improves the quality and supply of water for human consumption. Dam construction and other channel modifications, siltation, and industrial and agricultural pollution have all degraded the river habitats on which most species depend. As a result, the species richness and the abundance of freshwater snails have declined. Likewise, anthropogenic activities around the reservoir are on the increase, further compounding the problems and effects of pollution on the biota living within the aquatic habitat.

Shiroro Lake was constructed for domestic consumption within the Shiroro Local Government Area of Niger State and its environment. But there is little or no information about the snail’s distribution, diversity, and some selected physico-chemical parameters. This present research would serve as baseline information on the distribution, diversity of snails and changes in some physico-chemical parameters of the lake.

Materials and Methods

Description of the Study Area

Shiroro Lake was created with major objective of providing domestic water to Shiroro Local Government and its environment; however, fishing and irrigation have become other established uses of the reservoir. The area has a tropical climate with mean annual temperature, relative humidity and rainfall. The climate presents two distinct seasons: a rainy season and a dry season. The vegetation in the area is typically grass dominated savanna with scattered trees. The people are mostly engaged in agriculture, trading, artisanship and civil service for their living. Shiroro Lake was created in May, 1984 by damming the Kaduna River at Shiroro village, Niger State, Nigeria. Its coordinate is: Latitude 9.9724, Longitude 6.83532. The reservoir has an estimated surface area of 312 km² and a mean depth of 22.4 meters and continues to grow. It is now the second largest man-made lake in Nigeria followed by Jebba. The Shiroro Lake, like most other large man-made lakes in Nigeria and throughout the tropics, was expected to provide favourable conditions for large scale fish production and fishery development in Nigeria.

Water Sampling

Monthly sampling of the three study stations were carried-out from March to August, 2017 during the period of wet and dry seasons. Water samples were collected using 1 L plastic container from three sampling sites and transported to the Laboratory of the Department of Water, Aquaculture and Fishery Technology (WAFT), Federal University of Technology, Minna. Parameters like temperature, total dissolved solid, dissolved oxygen, pH and electrical conductivity were measured immediately from the sampling sites (Figure 1).

Figure 1: Map of the sampling stations of the Shiroro Lake, Niger State, Nigeria.

Snails Collection

Snails were collected from the study areas in the three sampling sites with the use of scoop net. Collected snails were transferred to a 1 L well labeled sample bottle with a cork and was preserved in 10% formalin. The samples were taken to the laboratory, viewed with the hand lens and subsequently identified using modified keys as described by [4] and pictorial diagrams.

Physico-chemical Parameters Analyses

Water Temperature

Water temperature at various sites were determined using mercury-in-glass thermometer which was immersed in water 6 cm below the water surface and left to stabilize for about two-five minutes before the readings were taken and recorded in °C.

DO, BOD, TDS, pH and EC Determination

Dissolved oxygen was determined with Hanna instrument (Hanna microprocessor pH /EC/TDS, P.R. (1970). This was done in duplicate for each site and each month of the sampling periods. The BOD was also determined using Hanna instrument after the samples had been incubated in the dark at laboratory temperature for five days. These were measured in mg/L. The Total Dissolved Solids (TDS in mg/L) and Electrical Conductivity (EC in µS/cm) were determined by the same instrument after rinsing in distilled water each time. The probe meter was standardized with buffer solutions of pH 4.0, 7.0, 9.0 before taking the reading for the pH in each station.

Sodium

Stock sodium was prepared by measuring 2.542 g of dried NaCl which was then diluted with a litre of distilled water. Intermediate sodium solution was prepared from stock solution by diluting 10 ml of stock solution with 100 ml distilled water. Standard sodium solution was then prepared from the intermediate sodium solution by diluting 10 ml of intermediate sodium solution with 100 ml distilled water. This was then used to prepare various concentrations in the range of 0.1 to 1.0 mg/L. The emission of these various concentrations was determined with a flame photometer at 589 nm. The Na content of the water sample was determined by measuring their emission with the flame photometer.

Potassium

Stock solution was prepared by dissolving 1.907 g of dried KCl crystal in distilled water which was then made up to 1 litre. Intermediate potassium solution was prepared by diluting 10 ml of stock potassium solution with 100 ml distilled water. Standard potassium solution was prepared by mixing 10 ml of intermediate potassium with 100 ml distilled water. Various concentrations in the range of 0.1 to 1.0 mg/L were prepared from the standard and were measured with flame photometer at 768 nm.

Nitrate

The nitrate concentration in the sampled water was determined using phenol disulphonic acid method. This method was carried out using spectrophotometer, laboratory glassware, hot water bath and reagents. Phenol disulphonic acid; 25 g of white phenol was dissolved in 150 ml (concentrated) and 85 ml of concentrated sulphuric acid was further added. The solution was heated until it dried out. 25 ml of water sample was placed in a porcelain basin and was evaporated to dryness on a hot water bath. 0.5 ml of phenol disulphonic acid (reagent 1) was then added to the residue and stirred with glass spatula. 5 ml of distilled water was added and 1.5 ml of potassium hydroxide solution (reagent 2) was also added. The mixture was thoroughly stirred by mixing. It turned yellow indicating the presence of Nitrate. The absorbance was read using spectrophotometer at 410 nm [5].

Phosphate

The concentration of phosphate in the water sample was determined by placing 25 ml of water sample in an Erlenmeyer flask and evaporated to dryness. The residue was cooled and dissolved in 1 ml of 70% perchloric acid (reagent 1). The flask was heated gently until the contents became colourless. It was cooled and 10 ml of distilled water was added together with 2 drops of Phenolphtalein indicator (reagent 2)_ this was prepared from 1.0 g of phenolphthalein dissolved in 100 ml of ethyl alcohol and 100 ml of distilled water. The above was titrated against sodium hydroxide solution (reagent 3) which was prepared by dissolving 4.0 g of the sodium hydroxide in 100 ml distilled water until there was an appearance of a slight pink colour. Volume was made up to 25 ml by adding distilled water; 1 ml of ammonium molybdate solution was added (reagent 4 made from 62 ml concentrated sulphuric acid and 80 ml distilled water and allowed to cool. 5 g of ammonium molybdate was then dissolved separately in 35 ml of distilled water and mixed with the sulphuric acid solution to 200 ml). 3 drops of stannous chloride solution (reagent 5_this was made from 0.5 g stannous chloride and dissolved in 2 ml hydrochloric acid and then, diluted to 20 ml with distilled water and used fresh). A blue colour indicates the presence of phosphate. The absorbance was recorded on spectrophotometer 690 nm after 10 minutes.

Data Analysis

One way Analysis of Variance (ANOVA) was used to determine the monthly variations in physico-chemical parameters using SPSS IBM (Version 20 for window) statistical package at P<0.05 level of significance. Taxa richness (Margalef and Menhnick indices), Diversity (Shannon and Simpson dominance indices), Eveness indices and Huctchenson T-test for inter-site comparison were determined using the computer basic programme SP DIVERS.

Results and Discussions

Monthly Variation in Physico-chemical Parameters of the Sampling Stations of Shiroro Lake, Minna Niger State

Water Temperature

The highest water temperature was recorded in station 1 with 30.5°C in the month of May and the lowest was recorded at station 1 in the month of August (Table 1).

DO. The highest dissolved oxygen was recorded in station 1 with 5.4 mg/L in the month of March and the lowest was recorded at station 2 in the month of August (Table 1).

BOD. The highest BOD was recorded at station 1 with 5.2 mg/L in the month of March and the lowest was recorded at station 2 in the month of August (Table 1).

TDS. The highest TDS was recorded in station 1 with 108 mg/L in the month of June and the lowest was recorded at stations 2 and 3 in the month of August (Table 1).

pH. The highest pH was recorded in station 1 with 10 in the month of March and the lowest was recorded at station 3 in the month of August (Table 1).

Electrical Conductivity. The Electrical conductivity for the three stations was the same through-out except for the Electrical conductivity of station 1 which increased slightly from 0.03 to 0.04 µS/cm in June (Table 1).

Sodium. The highest Sodium concentration was recorded in station 1 with 8.83 mg/L in the month of July and the lowest was also recorded at station 1 in the month of May (Table 1).

Potassium. The highest potassium concentration was recorded in station 3 with 4.11 mg/L in the month of March and the lowest was recorded at station 2 in the month of April (Table 1).

NO₃. The highest Nitrate concentration was recorded in station 3 with 0.83 mg/L in the month of July and the lowest was recorded at station 2 in the month of March (Table 1).

PO₄. The highest phosphate concentration was recorded in station 3 with 4.11 mg/L in the month of March and the lowest was recorded at station 2 in the month of April (Table 1).

Table 1: Summary of the physico- chemical parameters of the study stations in Shiroro Lake, Niger State from March to August, 2017. The highest and lowest values obtained during the sampling periods are indicated in parenthesis.

Abundance and diversity of snail species collected from Shiroro Lake. Total number of 7 species of snails was encountered during the study period. 2 species of Melanoides polymorpha were found in stations 2 and 3 but none in station 1. Ballamya phthinotropis was the major species with relative aboundance of 413 species (37%) followed by Ballamya capillata with 346 species (31%), Afrogyrus rodriguezensis with the total number of 178 species (16%) and Melanoides polymorpha with the lowest number of species (0.4%) (Table 2). Shannon index and Eveness index analyses showed that there were significant differences (P< 0.05) among the stations (Table 3).

Table 2: Spatial variations in snail species collected from Shiroro Lake, Niger State from March to August, 2017.

Table 3: Taxa richness, Diversity, Eveness and Dominance Indices of Snail species collected from Shiroro Lake, Niger State from March to August, 2017.

Taxa-S, Dominance_ D, Simpson_1-D, Shannon_H and Eveness_e^H/S stand for Taxa richness, Dominance Diversity, Simpson indices, Shannon indices and Eveness indices respectively.

Discussion

During the dry season the water temperature recorded was high in March, April, and May, 2017. This may be due to the increase in solar radiation (which is a usual phenomenon during dry season) during the period of the study. The mean water temperature obtained in this study was typical of tropical inland fresh water and river. This is in line with the findings of [6]. This is also in agreement with the studies of [7], [8] and [9] who also recorded high temperatures during the dry season. In addition, the relatively low temperature recorded in the months of June, July, August, 2017 may be due to the onset of the raining season and increasing water volume. This finding is also in agreement with the reports of [7] and [8].

The mean dissolved oxygen values obtained during the period of the study for the stations were low. The low level of DO probably indicated polluted nature of the water body. Similar low level of DO was reported in Dal Lake, Kashmir [10]. Monthly variations in DO for all the three stations were fluctuating. This fluctuation in the levels of DO could be as a result of differential influx of pollutants as run-offs from the neighbouring communities. It could also be due to rainfall regime pattern.

The BOD values indicate the extent of organic pollution in the aquatic system which affects the water quality [10]. Based on the BOD classification of [5], the mean BOD values in station 1 was high (2.7 mg/L), while stations 2 and 3 were low (2.4 mg/L) and (2.2 mg/L), respectively compared to that of station 1. The fluctuations in the BOD of each station and month probably indicate that the water in these stations was moderately polluted. This may be due to anthropogenic activities around the lake and the presence of the market at the lake side which may have led to organic pollution.

The monthly variations in Nitrate level were relatively high in all the sampling stations with the highest value of 0.83 mg/L in the month of July. Slightly higher range of values was reported in the studies of [12] in Bahir Dar Gulf of lake Tana, Ethiopia and [13] in a perturbed Tropical Stream in the Niger Delta, Nigeria; and [9] in River Galma, Zaria with 0.92-4.18 mg/L, 0.22-2.87 mg/L, 0.03 ± 0.0-1.11 ± 0.04 mg/L, respectively). Furthermore, the mean phosphate concentration ranged from 0.24-0.42 mg/L. This is higher than the mean values of 0.01 ± 0.01-0.20 ± 0.01 mg/L reported by [9]. Lower levels of phosphates, sulphates, nitrates indicate low level of organic pollution. The highest sodium content was recorded in July with 8.83 mg/L. This is probably because the most common source of elevated sodium levels in the lake water are: erosion of salt deposit and sodium bearing rock minerals, infiltration of surface water contaminated by road salt, irrigation and precipitation of leachate from landfill or industrial sites [14]. And during the rainy season there was influx of run-offs from the surrounding environment into the water body.

The EC in this research was very low (0.03 μs/cm) and would probably not pose any threat to the biota of the lake. The FEPA acceptable limit for conductivity in domestic water supply is 70 μs/cm [15]. Higher values were recorded from the same study site by [16]. This is also in contrast to the findings of [9] who reported a range mean value of 69.20 ± 3.12-157.80 ± 24.69 μS/cm in River Galma, Zaria. Likewise, the TDS are very low in comparison with previous studies. This may have arisen from improved maintenance of the lake.

The snail species in Shiroro Lake is highly diverse with Bellamya phthinotropis dominating the Shiroro Lake. The numerical density and species richness of snails were higher in the raining season than in the dry season. The snail species may have responded to changes in the water quality parameters as it was observed in changes in composition of species assemblage and abundance in the various sites. They may also have came out of their hiding places since there are now increased vegetation cover that could portend improved feeding and fertile ground for reproduction. Similar studies by [17] reported 29.06% relative abundance of mollusc in Obazuwa Lake, Benin city and attributed the abundance of the molluscs and oligochaetes in all the stations to the non-occurrence of habitat restriction in the study areas.

Conclusions and Recommendations

Anthropogenic activities and changes in season influenced the environmental conditions of the Shiroro Lake, thus affecting the snail species composition in each station. Overall results showed that changes in water quality of the Lake have significant effects on the structure, abundance and diversity of the snail species that were found. Station 3 had the highest number (403), station 2 with 363 species and station 3 had the lowest with 345 species. The family Viviparidae and Bithyniidae has 2 species each, the Bellamya phthinotropis has the highest number of individual species followed by Bellamya capillata and the lowest was recorded among the species of Gebtella barthi. In general the abundance of Snail was higher during the rainy season than during the dry season.

The highest DO was observed in the month of March with 5.4 mg/L, the highest Electrical conductivity was observed in the month of April with 0.4 (µS/cm), the highest TDS was observed in the month of April with 108 mg/L, the highest BOD was observed in the month of March with 5.2 mg/L. The variations in the phosphate, nitrates, sodium and potassium indicate that Shiroro Lake is organically polluted but do not pose any serious danger to the survival and adaptation of the biota.

More care should be taken to minimize the entry of pollutants into the water body so that the snails species and other biota of the Lake observed in this water body can be conserved.

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