A Simple PCR Procedure to Detect White Spot Syndrome Virus (WSSV) of Shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis> (Fabricious)

A non-stop, single tube and semi-nested polymerase chain reaction (PCR) technique with simple procedure was developed for simultaneous detection and grading the level of white spot syndrome infection in penaeid shrimp, Penaeus monodon. In this PCR procedure, three sense primers and one antisense primer with uniform annealing temperature of 55 °C were used. These primers amplify three PCR products (500, 300 and 200 base pairs [bp]) depending upon the severity of infection. Quantities of WSSV-DNA at 10 pg and greater gave three PCR products of 500, 300, 200 bp. A moderate concentration of WSSV-DNA, around 100 fg, gave two products of 300 and 200 bp and a low concentration of 1 fg or more gave only one PCR product of 200 bp. This PCR technique was assessed for early detection of WSSV in shrimp. In time-course infectivity experiments conducted on shrimp with WSSV, one PCR product (200 bp) was seen in hemolymph, tail tissue and gill at 3 h post infection (p.i.); two PCR products (300 and 200 bp) were seen in tail tissue, hemolymph, heart tissue and gill at 18 h p.i. At 30 h p.i., three PCR products (500, 300, 200 bp) were seen in all the organs tested. The samples collected from control animals showed negative for WSSV.     

White spot syndrome virus (WSSV) infection in tiger shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>: A non-lethal histopathological rapid diagnostic method using paraffin and frozen sections

White spot syndrome virus (WSSV) infection was induced in tiger shrimp, Penaeus monodon, under laboratory conditions, and histopathological changes in subcuticular epithelial cells of the eye stalk and pleopod were studied sequentially at different time post-challenge. Routine histological techniques using paraffin embedded tissues, as well as frozen tissues, were used to document WSSV infection. Histological manifestations such as cellular hypertrophy in the subcuticular epithelial cells of the eyestalk and pleopod could be detected as early as 18 h post-infection (p.i.) before the manifestation of clinical signs of the disease. However, no histopathological changes could be detected before 18 h p.i.. Hypertrophy of the nuclei in the epithelial cells was pronounced after 24 h p.i. Marked necrosis, and eosinophilic intranuclear inclusions, characteristic of early stages of WSSV infection were observed between 24–36 h p.i. Clinical signs of the disease appeared at 48 h p.i. The presence of WSSV at early asymptomatic stages of p.i. has been tested in parallel samples using polymerase chain reaction, for further confirmation of WSSV. This paper discusses the potential of a non-lethal and rapid histopathological diagnostic method to document WSSV infection, using the eyestalk or pleopod, when expensive DNA based diagnostics are not available or affordable.     

Comparison of dot blot and PCR diagnostic techniques for detection of white spot syndrome virus in different tissues of Penaeus monodon

Comparison of PCR and dot blot diagnostic techniques for detection of white spot syndrome virus (WSSV) was made on different tissues of infected Penaeus monodon including eye stalk, eye stalk with eye, gills, cuticle, pleopod, periopods, uropods and telson. Dot blots of crude DNA extracted from infected tissue samples showed positive reactions with all the samples; however, the sensitivity of the dot blot was reduced with the purification of DNA samples extracted from pleopod, telson and uropod. PCR was found to be more sensitive when compared to dot blot. Both crude DNA and purified DNA samples extracted from all the tissues except for eye stalk with eye showed single step nested PCR positive reaction. The amplification of all or either of the three bands of 941 bp, 525 bp and 204 bp size varied with the tissues analysed. The severity of infection assessed by PCR amplification was found to be maximum in cuticle and telson followed by gill. Other tissues such as eye stalk, pleopod, periopods and uropod were observed to have mild infection. The maximum intensity of the PCR product was for the smallest amplified product of 204 bp followed by 525 bp and the weakest intensity was observed for the 941 bp size. The limitation of PCR due to inhibiting factors present in tissues could be overcome with the use of dot blot which gave positive reaction from the DNA extracted from eye stalk including the eye but yielded no amplification by PCR.     

Differential host passaging alters pathogenicity and induces genomic variation in white spot syndrome virus

To understand the pathological effects of passaging WSSV through different animals, we passaged WSSV through crabs (Portunus sanguinolentus and P. pelagicus), prawn (Macrobrachium rosenbergii) and artemia (Artemia sp.). Among the three geographically distinct WSSV isolates (A166, G9 and G27) that caused over 95% mortalities in experimental infections in P. monodon, only two isolates (A166 and G9) caused mortality (5%) in M. rosenbergii. On subsequent infection of P. monodon with the passage one virus (P1V) from M. rosenbergii, only P1V of A166 caused 5% mortality while P1V of G9 failed to cause any mortality, though the infected shrimp were positive for WSSV by nested PCR. Crabs showed differential susceptibility to WSSV isolates. While passaging WSSV (G9) through P. sanguinolentus retained pathogenicity, passaging through P. pelagicus completely attenuated the two strains (A117 and G27). Passaging through M. rosenbergii changed pathogenicity of isolates (A166 and G9) from 95% to 5% cumulative mortality in experimental infections. WSSV was unable to infect Artemia and could not transmit the disease to P. monodon. WSSV (A117) passaged through P. monodon retained its virulence. An examination of tandem repeats (TR) in ORF 94 of the WSSV genome revealed variations upon passaging through different hosts. One isolate (A117), with 12 TR yielded 8 TR when passaged through P. pelagicus and 18 TR when passaged through P. monodon. Two other isolates (G9 and G27) with 9 TR, retained them when passaged through P. monodon and P. sanguinolentus but yielded 8 and 12 TR when passaged through P. pelagicus and M. rosenbergii, respectively. Thus, differences in TR number appeared to result from host selection rather than geographical isolation.     

The effectiveness of commercial probiotics in northern white shrimp <Emphasis Type="Italic">Penaeus vannamei</Emphasis> ponds

ABSTRACT:   The effectiveness on water quality, population density of bacteria, and shrimp production in ponds treated with commercial probiotics was tested in Penaeus vannamei ponds in Hai-yan, China. Six ponds with replicates for treatment and control were used. Results showed that the probiotics could improve the population density of beneficial bacterial flora, reduce concentrations of nitrogen and phosphorus, and increase yields of shrimp. The average counts of Bacillus sp., ammonifying bacteria, and protein mineralizing bacteria were found to be significantly higher in treated ponds compared to control ponds ( P  < 0.05). In control ponds, an increase in presumptive vibrios was observed and the average density was up to 2.09 × 10³ cfu/mL, whereas that was only 4.37 × 10² cfu/mL in treated ponds ( P  < 0.05). The use of probiotics also significantly increased dissolved oxygen ( P  < 0.05) and reduced dissolved reactive-phosphorus, total inorganic nitrogen and chemical oxygen demand ( P  < 0.05). An average of 8215 ± 265 kg shrimp/ha was obtained in treated ponds with a feed conversion ratio (FCR) of 1.13 ± 0.05 and survival rate of 81.00 ± 6.25% compared with 4985 ± 503 kg shrimp/ha, 1.35 ± 0.12 and 48.67 ± 3.51%, respectively, in control ponds. This indicates that the addition of the commercial probiotics had a noticeable influence on water quality of shrimp ponds and shrimp production.     

TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan‐based real‐time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L^?1) for 10 days and challenged with WSSV by feeding WSSV‐positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post‐infection. The TaqMan real‐time PCR assay showed a highly dynamic detection limit that spanned over 6 log₁₀ concentrations of DNA and high reproducibility (standard deviation 0.33–1.42) and small correlation of variability (CV) (1.89–3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L^?1 of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L^?1 of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L^?1 (TAN) had increased susceptibility to WSSV.     

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) was developed and field testing was conducted. A 604‐bp target sequence was selected from the vp28 gene of WSSV. A primer set was developed to amplify a 338‐bp DNA fragment at the junction of the NS2 and NS1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139‐bp PCR fragment from the β‐actin gene by alignment of this gene from Litopenaeus vannamei, Fenneropenaeus chinensis and Penaeus monodon. The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV, WSSV and β‐actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170 L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV, and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus‐free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV.     

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L^?1 were subjected to salinities of 50 g L^?1, 35 g L^?1, 20 g L^?1, 10 g L^?1 and 7 g L^?1 or 5 g L^?1 and simultaneously exposed to 10^5.5 SID₅₀ mL^?1 of WSSV for 5 h, after which the salinity was brought back to 35 g L^?1. Shrimp that were transferred from 35 g L^?1 to 50 g L^?1, 35 g L^?1 and 20 g L^?1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L^?1 to 10 g L^?1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L^?1, 7 g L^?1 and 5 g L^?1, was 6.7%, 46.7% and 53.3%, respectively (P < 0.05)_. For testing the effect of moulting, shrimp in early premoult, moulting and post‐moult were immersed in sea water containing 10^5.5 SID₅₀ mL^?1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post‐moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L^?1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

Enhancing the dietary value of palm oil in the presence of lysolecithin in tiger shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis>

The effect of four iso-nitrogenous and iso-lipidic diets containing soy lecithin and lysolecithin with fish oil (sardine) and palm oil on growth, digestibility, and fatty acid composition of tail muscle and non-muscle portions of tiger shrimp, Penaeus monodon, was evaluated. Shrimp fed with lysolecithin diets had significantly (P?<?0.05) higher daily growth coefficient values (1.40–1.45% day^?1) than those fed with soylecithin containing diets (1.32–1.37% day^?1). Correspondingly, lysolecithin-supplemented diets showed significantly higher (P?<?0.05) apparent digestibility coefficients (ADC) of fatty acids with both the oils due to higher emulsification ability of small micelle forming by lysolecithin. However, there were no significant differences in survival and FCR among all treatments. The fatty acid composition of the test diets reflected to a certain extent in the fatty acid composition of the muscle and non-muscle portions of shrimp. Arachidonic, eicosapentaenoic, and docosahexaenoic acid contents of muscle and non-muscle portions of shrimp were significantly (P?<?0.05) higher in lysolecithin-supplemented diet compared to soylecithin-supplemented diet. The present results suggest that lysolecithin improved the fatty acid digestibility with its high emulsification properties that reflected in better performance by improving dietary value of palm oil.     

Enhancement of <Emphasis Type="Italic">Penaeus monodon</Emphasis> shrimp postlarvae growth and survival without water exchange using marine <Emphasis Type="Italic">Bacillus pumilus</Emphasis> and periphytic microalgae

We have investigated the possibility of using a consortium of marine bacterium and periphytic microalgae to improve the water quality and increase the growth and survival of the shrimp Penaeus monodon in a hatchery system. Three treatments were evaluated for their effect on P. monodon postlarvae (PL) when the culture water was not changed: Bacillus pumilus alone (B); periphytic microalgae alone (M); B. pumilus + periphytic microalgae (BM). P. monodon PL raised in a tank of unchanged water without bacterium and periphytic microalgae served as the control. The water in tanks of the M and BM treatments had significantly low levels of total ammonia-nitrogen (TAN) (0.03 and 0.01 mg l⁻¹, respectively) and nitrite-nitrogen (NO₂-N) (0.03, 0.01 mg l⁻¹, respectively) than that in the B (TAN 0.80, NO₂-N 0.68 mg l⁻¹) and control (TAN 1.11, NO₂-N 1.12 mg l⁻¹) tanks. Moreover, PL cultured in tanks M and BM had significantly higher survival and specific growth rates and a significantly higher resistance to the reverse salinity stress test than those in the B and control tanks. Compared to the control PL, the PL cultured in the BM tanks had significantly higher levels of protein, lipid, polyunsaturated fatty acids, ecosapentaenoic acid, and docosahexaenoic acid. The culture water in tanks BM also contained significantly less Vibrio than the control water. Our results illustrate the beneficial effects of a B. pumilus and periphytic microalgae consortium on improving the water quality and the growth and survival of shrimp PL grown in a hatchery system.     

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 10⁵ viral DNA copies.     

Detailed monitoring of white spot syndrome virus (WSSV) in shrimp commercial ponds in Sinaloa, Mexico by nested PCR

WSSV has caused great losses to the global shrimp industry in recent years. This virus can infect shrimps asymptomatically. However, once the clinical signs are developed, mortalities can reach 100% in 3-10 days. PCR has been extensively used to detect WSSV in a specific and sensitive manner. Nested PCR is even more sensitive than single-step PCR and had been used for the detection of WSSV in asymptomatic populations. In this work, a detailed monitoring of WSSV by nested PCR in shrimp commercial ponds in Guasave County, State of Sinaloa, Mexico, is presented. Five ponds from two different farms were monitored for growth and presence of WSSV. At the beginning of the culture, ponds from both farms showed no or very slight WSSV presence. A 3-day period of rain occurred at both farms 10 and 14 weeks of culture for farms 1 and 2, respectively. At this time, WSSV was widely distributed in the shrimp populations of farm 1 according to nested-PCR data, although no visual symptoms were observed. In ponds of farm 2, WSSV was present at low level. However, the number of PCR-positive groups was drastically increased in both farms by nested and single-step PCR. Abrupt fluctuations in temperature and salinity were documented in farm 2 after the rain, which may have contributed to the increasing of viral load in the pond's shrimp populations. Twelve days after the rain period, estimated mortalities of 80% occurred in farm 1. Nevertheless, the study ponds at farm 2 culture continued normally for three more weeks and were harvested successfully (52% and 67% of survival for ponds 1 and 2, respectively). The removal of 40% and 50% of shrimp population 2-4 days after the raining period may have contributed to the thriving of the cultures. Analyses of the presence of WSSV in individuals of both sexes indicated that there is no preference for this virus to infect male or female shrimp. Also, no differences in weight were found between WSSV infected and non-infected individual shrimps, as well as nested-PCR positive against single-step PCR positive organisms. Nested PCR is more useful to monitor shrimp cultures than single-step PCR since it allows knowing how widely distributed the virus is in asymptomatically populations.     

Compensatory growth of Chinese shrimp, <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> following hypoxic exposure

Compensatory growth following stress is a strategy aquatic animals use to adjust themselves to a variable environment. Studies on the recovery growth of aquatic animals are not only of theoretical value in ecophysiology and evolution, but also important to applications in aquaculture and fisheries resource management. In this experiment, juvenile Fenneropenaeus chinensis with an initial average body weight of about 3.72 g were exposed to hypoxic water (about 2.08 and 3.11 mg/l of dissolved oxygen (DO) content) for 10 days and then switched to normoxic water (about 5.63 and 5.59 mg/l DO). Compared with juveniles in normoxia, juveniles in the hypoxia period allocated a greater proportion of energy to metabolism and exuviations but allocated less energy to daily metabolism per gram shrimp weight (J/g/day). This reduced feed conversion efficiency and feeding rate. Finally, F. chinensis suffered growth depression. The juveniles completely compensated for hypoxia-induced growth depression in 30 days after being switched into normoxic water and the compensation was achieved mainly by hyperphagia and slightly by improvement of feed conversion efficiency. During the recovery period the hypoxic-stressed shrimp showed higher daily metabolic energy (J/g/day) than controls (P < 0.05). Which means the stressed shrimp had more energy for feeding-related activities. So hyperphagia was observed. Energy analysis indicated that F. chinensis improved feed-conversion efficiency mainly by reducing the percentage of energy lost in feces and exuviations. The results showed that short-term non-lethal hypoxia would not affect the growth of juvenile F. chinensis if there were enough time for the stressed shrimp to recover. This suggested F. chinensis was capable of adapting to DO fluctuation to some extent and short-term non-lethal hypoxia would not have an obvious effect on natural, released, and cultured shrimp stock.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

The incidence of suspected white spot syndrome virus in semi‐intensive and extensive shrimp farms in Bangladesh: implications for management

The study was conducted to assess key factors influencing suspected white spot syndrome virus (WSSV) disease and associated shrimp production and economic performance in three contrasting black tiger shrimp (Penaeus monodon) culture technologies promoted by the United States Agency for International Development funded Shrimp Quality Support Project (SQSP) in Bangladesh. A total of 350 traditional, 315 Modified Traditional Technology1 (MTT1), 36 MTT2 and 88 Closed System Technology (CST) farmers from 10 sub‐districts in three districts of Khulna division were surveyed following random sampling at the end of the project. Binomial probit regression analysis revealed that smaller newly constructed ponds (known locally as gher) were less susceptible to WSSV, provided aquatic weeds were controlled using chemicals. Removal of sludge from ghers also had a positive effect, irrespective of technology and location. It was also shown that stocking of screened shrimp postlarvae (PL) does not guarantee protection against WSSV (t = 1.39, P > 0.05). Higher shrimp production was obtained by farmers practicing CST, followed by those operating MTTs and traditional technology respectively. Farmers who adopted CST also gained higher profitability followed by those operating MTT1, MTT2 and traditional technology.