Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 01‐08 ISSN 1023‐6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Functional response of Phytoseiulus persimilis Athias-Henriot to Tetranychus urticae Koch: effects of prey life stages and temperature Md. Fazlul Hoque, Md. Wahedul Islam and M. Khalequzzaman* Institute of Biological Sciences, University of Rajshahi, Rajshahi 6205, Bangladesh *Corresponding author email: [email protected]
Abstract: This paper examines the effect of density and temperature on the functional response of adult female Phytoseiulus persimilis to different life stages of the spider mite, Tetranychus urticae. The functional response was determined using functional response assay technique with plant leaf discs an arena. We conducted a logistic regression of the proportion of prey consumed as a function of initial prey density to identify functional response types, and used nonlinear least- squares regression and the random predator equation to estimate attack rates and handling times. In all the cases number of adult male prey attacked by female predator were greater than adult female prey. P. persimilis consumed more eggs and larvae than other stages of prey. Female predator consumed adult prey at each temperature which generally increased with prey density initially but leveled off at higher prey densities. The highest number of eggs, larvae and nymphs consumed by predator in 24 hours was 16.3 ± 0.42, 9.9 ± 0.59 and 9.5 ± 0.40 respectively whereas the highest number of eggs, larvae and nymphs consumed by a single female in 48 hours was 19.5 ± 1.15, 18.6 ± 0.62 and 13.3 ± 0.65 respectively. Handling time generally decreased with temperature whereas successful attack rate increased with temperature except at 30º C. Judging by a/Th values, P. persimilis was most efficient against T. urticae at 30-35ºC, about half as efficient at 25 ºC and performed poorly at 20º C. Again, from a/Th values, P. persimilis was most efficient against T. urticae in egg stage, about half as efficient at larva and nymph stages and performed poorly at adult stage.
Key words: Phytoseiulus persimilis, Tetranychus urticae, functional response, predation, temperature.
Introduction most predator-prey relationships are complex, the functional response of predators, or the The dynamics of predators and their prey is one of relationship between the number of prey the more extensively studied areas of ecological consumed per predator and prey density dynamics. This is particularly true for insects, due (Solomon, 1949; Holling, 1959), is basic to all to their significant impact on global food predator prey interactions (Hassell, 1978). production and human health and because their Complications in the interpretation of predator short life cycles and small size make them highly responses may arise because functional response amenable to laboratory study (Taylor, 1984). It is can be influenced by a number of factors, widely believed that in most animal species, including: (1) characteristics of predator and prey population growth rate is a decreasing function of (Abrams, 1990), (2) characteristics of prey, and density (Turchin, 1995). Thus, per capita growth (3) attributes of an environment (Huffaker et al., rate is negatively associated with present and/or 1970). past population densities (negative density dependence). Phytophagous insect populations Understanding predator prey interactions is crucial exist at relatively low to extremely high density. if a biological control programme is to be Top-down factors (i.e. natural enemies) can act to successful. Determining the effects of predation depress herbivore population size, usually well on prey populations is most commonly done below the host-plant’s carrying capacity through the analysis of functional and numerical (Cappuccino, 1992; Hunter & Elkinton, 1999). responses (Huffaker & Messenger, 1976). The Alternatively, bottom-up factors, such as plant number of prey attacked can then be used to help defenses, can control herbivore population growth predict predator development, survival, and (Hunter & Price, 1992). reproduction (Murdoch & Oaten, 1975). Functional response, as originally described by Solomon The role of predators in regulating or suppressing (1949), is the change in prey number killed per prey populations has been studied extensively by individual predator per unit time as a function of population biologists. Although the dynamics of 2 Haque et al.
changes in prey density. Early functional response prey had been consumed. Temperature was research was conducted by Holling (1959). He recorded hourly. The number of preys consumed formulated the mathematical models to describe by predators at each density were recorded after predatory responses that were influenced by 24 and 48 hours respectively from the starting of changes in predator behaviour. the experiment. Sets of experiments were repeated at 20°, 25°, 30° and 35°C at prey Holling (1959, 1961,1965) recognized three types densities of 10, 20, 30, 40, 50 and 60 maintained of functional responses: (1) a linear rise to a in incubators. plateau, typical of filter feeders (type I and II), (2) a curvilinear rise to a plateau (with negatively In this work formulae of type I and II models of accelerating rate), characteristic of invertebrate Holling (1959 ) and type III model of Hassel et al. predators (type II and III), and (3) a sigmoid curve (1977) were used as below: also reaching a plateau (with a positive Type I : N = a`TN accelerating rate up to the inflection point and a thereafter a diminishing rate up to the plateau), a'TN typical of those predators with a search image Type II : Na = (type III). Mori & Chant (1966) found a dome- 1+ h Na'T shaped response (type IV) for Phytoseiulus Type III : N = N[1–exp(–a`[T–T N ])] persimilis Athias-Henriot attacking Tetranychus a h a urticae Koch. This paper presents the results of a Where Na is the number of prey killed, N is the set of experiments examining how the predatory prey density, T is the time available for searching efficiency of P. persimilis feeding on different life during the experiments, a` is the search rate and stages of T. urticae, as measured by a functional Th is the amount of time the predator handles for response, was influenced by prey density and each prey killed. Data were analyzed using least temperature. square regression by fitting a line through the data point as predicted by the descriptive functional Materials and Methods response equation (Fuji et al., 1986) T. urticae colonies were maintained on bean leaf which was cultivated in the experimental fields of Tt Y = the Institute of Biological Sciences, University of + cXXat )]exp('/[1 Rajshahi. Experimental arenas were constructed h . by placing excised leaf disk from uninfested bean In this equation Y is the number of attacks per plants placed on petridish (12cm diameter) filled predator, Tt is the total time prey is exposed, X is with watery cotton bed in the laboratory. the prey density, a` is the search rate and th is the The eggs, larvae, nymphs and adult males and amount of time the predator handles for each prey females were placed on leaf disks at densities of killed and c is the facilitation coefficient, which 10, 20, 30, 40, 50 and 60 per disk. Adult T. urticae determines the form of the functional response. were transferred directly to arenas from the When c = a`.th, the functional response is Pseudo colonies on the day of the experiments using a Type I, c = 0 gives a Type II response, for c > fine point brush. Eggs were obtained by placing T. a`.th, the response is type III (Fuji et al., 1986). urticae adults on pest free bean leaves inside a The data were fitted to the descriptive equation petridish for two days prior to the experiments. On using the LAB Fit Curve Fitting Software by Wilton the day of the experiment egg were transferred and Cleide P Silva ver 7.2.42 (1999-2008). from the bean leaves inside the petridish to the Results arenas. Ten replications of each prey density were deployed for the female predator. The The daily average number of prey consumed by arenas were left undisturbed for at least one hour. the P. persimilis females at various prey stages They were then examined to determine if any and densities of T. urticae is shown in Table 1. In eggs had hatched or adults had laid eggs. One P. case of all developmental stages of prey generally persimilis was placed at the centre of each of the prey density played a significant role in predation leaf disks and allowed to feed for eight hours. of P. persimilis. The average number of preys After eight hours, the number of adult T. urticae consumed by P. persimilis females feeding on and cadavers were counted in the adult and larval eggs, larvae or nymphs of T. urticae gradually arenas, and the number of viable egg were increased at an accelerated rate when 10 to 60 counted in the egg arenas to determine how much preys were provided (Tables 1 and 2). Functional response of P. persimilis to T. utricae 3
Functional responses in all stages of prey in 24 increased with prey density initially but it was and 48 hours approximated Holling type II model leveled off at higher prey densities (Table 3). and estimated parameters of functional responses Functional responses at all temperatures are shown in Tables 1 and 2. Handling time approximated Holling type II model and estimated generally increased from egg to adult. Successful parameters of functional responses are shown in attack rate was the highest in the egg stage while Table 3. Handling time generally decreased with it was the lowest in the adult (♀) stage. Judging by the increase in temperature, whereas successful a/Th values, P. persimilis was most efficient attack rate increased with temperature except at against T. urticae in egg stages, about half as 30º C. Judging by a/Th values, P. persimilis was efficient at larvae and nymphal stages and most efficient against T. urticae at 30-35ºC, about performed poorly at the adult stage. The observed half as efficient at 25ºC and performed poorly at and predicted curves of functional response at 20º C. The observed and predicted curves of different densities at different prey stages in 24 functional response at different densities at and 48 hours are presented in Figs. 1 - 10. different temperatures are presented in Figs. 11- 14. The number of female preys consumed by female predators at each temperature generally
Table 1. Functional response parameters of P. persimilis (♀) to T. urticae in 24 hours Number of prey consumed in the form (mean ± S.E) Prey Density Egg Larva Nymph Adult (♂) Adult (♀) 10 4.8 ± 0.42 2.2 ± 0.20 2.1 ± 0.23 2.2 ± 0.33 1.1 ± 0.18 20 8.9 ± 0.50 4.4 ± 0.27 5.4 ± 0.48 4.5 ± 0.45 1.2 ± 0.20 30 12.8 ± 0.57 5.4 ± 0.31 4.6 ± 0.27 5.2 ± 0.33 1.3 ± 0.26 40 10.7 ± 0.56 6.6 ± 0.43 7.2 ± 0.55 5.4 ± 0.48 2.5 ± 0.27 50 14.4 ± 0.69 9.9 ± 0.59 8.7 ± 0.30 7.7 ± 0.47 2.6 ± 0.31 60 16.3 ± 0.42 9.7 ± 0.37 9.5 ± 0.40 7.4 ± 0.43 2.6 ± 0.27 a' 0.027 0.009 0.010 0.012 0.0036 th 0.958 0.580 0.924 1.75 4.16 a'/Th 0.0281 0.0155 0.0108 0.00685 0.000858 c 0.000948 0.0000218 -0.000153 -0.000405 0.00001625 R2 0.993 0.954 0.920 0.943 0.821 027.0 Nt 009.0 Nt 01.0 Nt 012.0 Nt 0036.0 Nt Na + 0258.01 Nt + 00522.01 Nt + 00924.01 Nt + 021.01 Nt + 0149.01 Nt *Na = number of preys consumed; Nt = number of preys available; Th = handling time; a’ = successful attack rate; c = facilitation coefficient.
Table 2. Functional response parameters of P. persimilis (♀) to T. urticae in 48 hours Number of prey consumed in the form (mean ± S.E) Prey Density Egg Larva Nymph Adult (♂) Adult (♀) 10 6.4 ± 0.43 3.4 ± 0.31 3.4 ± 0.37 2.5 ± 0.45 1.2 ± 0.20 20 10.9 ± 0.66 7.8 ± 0.47 7.4 ± 0.27 4.4 ± 0.31 1.3 ± 0.26 30 13.7 ± 0.37 12.4 ± 0.75 8.8 ± 0.42 6.4 ± 0.37 2.8 ± 0.33 40 15.8 ± 1.30 10.4 ± 0.62 8.4 ± 0.50 7.6 ± 0.50 2.6 ± 0.27 50 19.5 ± 1.15 18.6 ± 0.62 13.3 ± 0.65 8.2 ± 0.55 3.2 ± 0.33 60 19.4 ± 1.25 18.3 ± 1.19 12.9 ± 0.86 10.2 ± 0.42 3.8 ± 0.39 a' 0.016 0.009 0.0089 0.0057 0.0022 th 1.561 0.614 1.784 1.935 5.564 a'/th 0.0102 0.0146 0.00498 0.00293 0.000397 c 0.003306 -0.000058 -0.000156 -0.000684 0.000073 R2 0.984 0.892 0.895 0.986 0.902 016.0 Nt 009.0 Nt 0089.0 Nt 0057.0 Nt 0022.0 Nt Na + 02497.01 Nt + 00552.01 Nt + 01587.01 Nt + 01103.01 Nt + 0122408.01 Nt *Na = number of preys consumed; Nt = number of preys available; Th = handling time; a’ = successful attack rate; c = facilitation coefficient. 4 Haque et al.
Table 3. Functional response parameters of P. persimilis (♀) to T. urticae (♀) at different temperatures. Number of prey consumed at temperature (mean±S.E) Prey Density 20°C 25°C 30°C 35°C 10 1.3 ± 0.15 1.7 ± 0.26 2.1 ± 0.23 2.2 ± 0.29 20 1.4 ± 0.16 2.0 ± 0.39 2.2 ± 0.29 2.4 ± 0.31 30 1.5 ± 0.22 2.1 ± 0.31 4.2 ± 0.25 3.1 ± 0.28 40 2.7 ± 0.30 3.1 ± 0.28 3.2 ± 0.20 4.1 ± 0.31 50 3.2 ± 0.33 4.1 ± 0.31 4.8 ± 0.33 5.1 ± 0.41 60 3.1 ± 0.38 4.0 ± 0.26 5.0 ± 0.37 5.0 ± 0.47 a' 0.004 0.005 0.009 0.008 th 3.110 2.907 3.030 2.563 a'/th 0.001238 0.00171 0.00297 0.003055 c -0.00037 0.000254 -0.0000759 -0.0001 R2 0.840 0.860 0.777 0.904 004.0 Nt 005.0 Nt 009.0 Nt 008.0 Nt Na + 01244.01 Nt + 01453.01 Nt + 02727.01 Nt + 02050.01 Nt *Na = number of preys consumed; Nt = number of preys available; Th = handling time; a’ = successful attack rate; c = facilitation coefficient.
Egg Larva 18 12 16
14 10
12 8 10 6 8
6 4 Prey Consumed Prey Consumed 4 2 2 Observed Predicted Observed Predicted 0 0 0 10203040506070 0 10203040506070 Prey density Prey density Fig. 1. The functional response of P. persimilis (♀) to Fig. 3. The functional response of P. persimilis (♀) to eggs of T. urticae in 24 hours. Handling time (Th)= larvae of T. urticae in 24 hours. Handling time (Th) = 0.958 hr/egg, successful attack rate (a`) = 0.027 and 0.580 hr/larva, successful attack rate (a`) = 0.009 and c=0.000948 (χ2= 0.768 at 5df). c= 0.0000218 (χ2= 0.301 at 5df).
Egg Larva 25 25
20 20
15 15
10 10 Prey Consumed Prey Consumed 5 5 Observed Predicted 0 Observed Predicted 0 10203040506070 0 Prey density 0 10203040506070 Prey density
Fig. 2. The functional response of P. persimilis (♀) to eggs of T. urticae in 48 hours. Handling time (Th) = Fig. 4. The functional response of P. persimilis (♀) to 1.561 hr/egg, successful attack rate (a`) = 0.016 and larvae of T. urticae in 48 hours. Handling time (Th) = c=0.00331 (χ2 = 0.480 at 5df). 0.614 hr/larva, successful attack rate (a`) = 0.009 and c= -0.000058 (χ2= 1.496 at 5df). Functional response of P. persimilis to T. utricae 5
Nymph 12 Adult male 12
10 10
8 8
6 6
4 Prey Consumed
Prey Consumed 4
2 2 Observed Predicted
Observed Predicted 0 0 10203040506070 0 0 10203040506070 Prey density
Prey density Fig. 8. The functional response of P. persimilis (♀) to Fig. 5. The functional response of P. persimilis (♀) to adults (♂) of T. urticae in 48 hours. Handling time (Th)= nymphs of T. urticae in 24 hours. Handling time (Th) = 1.935 hr/adult (♂), successful attack rate (a`) = 0.00568 0.924 hr/nymph, successful attack rate (a`) = 0.010 and and c= -0.000684 (χ2=0.064 at 5df). c= -0.000153 (χ2= 0.630 at 5df). Adult Female Nymph 3 16
14 2.5
12 2 10
8 1.5
6 1 Prey Consumed Prey Consumed 4 0.5 2 Observed Predicted Observed Predicted 0 0 0 10203040506070 0 10203040506070 Prey density Prey density
Fig. 6. The functional response of P. persimilis (♀) to Fig. 9. The functional response of P. persimilis (♀) to nymphs of T. urticae in 48 hours. Handling time (Th) = adults of T. urticae in 24 hours. Handling time (Th) = 1.784 hr/nymph, successful attack rate (a`) = 0.009 and 4.16 hr/adult (♀), successful attack rate (a`) = 0.00357 2 c= -0.000156 (χ = 0.722 at 5df). and c= 0.0000163 (χ2 = 0.362 at 5df).
Adult male Adult female 9 4.5
8 4
7 3.5
6 3
2.5 5 2 4
Prey Consumed 1.5 3 Prey Consumed 1 2 0.5 1 Observed Predicted Observed Predicted 0 0 0 10203040506070 0 10203040506070 Prey density Prey density
Fig. 10. The functional response of P. persimilis (♀) to Fig. 7. The functional response of P. persimilis (♀) to adults (♀) of T. urticae in 48 hours. Handling time adults (♂) of T. urticae in 24 hours. Handling time (T ) = h (Th)=5.564 hr/adult(♀), successful attack rate 1.75 hr/ adult (♂), successful attack rate (a`) = 0.012 (a`)=0.0022 and c = 0.000073 (χ2= 0.290 at 5df). and c= -0.000405 (χ2 = 0.265 at 5df). 6 Haque et al.
20º C 35º C
6 4 3.5 5 3 4 2.5 2 3
1.5 2 1 Prey Consumed Prey Consumed 0.5 1 Observed Predicted Observed Predicted 0 0 0 10203040506070 0 10203040506070 Prey density Prey density
Fig. 11. The functional response of P. persimilis (♀) to Fig. 14. The functional response of P. persimilis (♀) to T. urticae (♀) at 20º C. Handling time (Th) = 3.110 T. urticae (♀) at 35º C. Handling time (Th) = 2.563 hr/adult, successful attack rate (a`) = 0.004 and c=- 2 hr/adult, successful attack rate (a`) = 0.008 and c = - 0.00037 (χ = 0.441 at 5df). 0.0001 (χ2 = 0.363 at 5df).
25º C Discussion According to Eveleigh and Chant (1981) the 5 functional response of protonymphs and 4 deutonymphs of P. persimilis to varying densities 3 of protonymphs of the Pacific spider mite, 2 T. pacificus under laboratory conditions was type II. Takafuji & Chant (1976) observed a linear rise
Prey Consumed 1 Observed Predicted to a plateau (type I) for protonymphs and 0 0 10203040506070 deutonymphs of P. persimilis attacking Prey density T. pacificus eggs. The linear rise to a plateau indicated that, as prey density increased, the searching efficiency of P. persimilis immature Fig. 12. The functional response of P. persimilis (♀) to stages was not affected by either the hunger level T. urticae (♀) at 25º C. Handling time (Th) = 2.907 of the predator and/or by the rise in the handling hr/adult, successful attack rate (a`) = .005 and c = 2 time of the prey by the predator (Holling, 1959, 0.000254 (χ = 0.603 at 5df). 1961; Fujii et al., 1986). According to Rasmy et al. (1991), at 22–23°C and 70% RH, protonymphs 30º C and deutonymphs of P. persimilis consumed 5.6 6 and 5.8 nymphal individuals of a mixed diet of 5 T. urticae and E. dioscordis respectively. The 4 functional responses of protonymphs and 3 deutonymphs of P. persimilis feeding on the egg 2 stage of T. urticae on bean leaves at 20°C and
Prey Consumed 1 Observed Predicted 60% RH were also type II (Fernando & Hassell, 0 1980). According to these authors, who studied 0 10203040506070 the functional response at 8 and 24 h periods, the Prey density plateaus of the functional response curves were reached at approximately 2.8 and 6 prey eggs per Fig. 13. The functional response of P. persimilis (♀) to predator protonymph, at both the initial prey T. urticae (♀) at 30º C. Handling time (Th) = 3.030 density of 20 eggs per arena for 8 and 24 h hr/adult, successful attack rate (a`) = 0.009 and c = - exposure times, respectively, whereas in the case 2 0.0000759 (χ = 0.536 at 5df). of the predator deutonymphs, these plateaus were observed at approximately 6 (at initial prey eggs of 20) and 20 (at initial prey eggs of 60) T. urticae eggs for 8 and 24 h experimental periods, respectively. Functional response of P. persimilis to T. utricae 7
All stages of T. urticae were consumed by P. References persimilis. As the density of juvenile prey Abrams, P.A. 1990. The effects of adaptive behavior on increased, predation also increased. The same the type-2 functional response. Ecology 71:877-885. trend was observed with the same species fed with T. cinnabarinus (Boisduval) (Friese & Cappuccino, N. 1992. The nature of population stability Gilstrap, 1982) or Panonychus ulmi Koch (Jolly, in Eurosta solidaginis, a non-outbreaking herbivore 2000). Other predatory mites showed the same of goldenrod. Ecology 73:1792-1801. type of functional response (Zhang et al., 1999; Castagnoli, M. & Simoni, S. 2004. Neoseiulus Thongtab et al., 2001; Castagnoli & Simoni, californicus (McGregor) (Acari: Phytoseiidae): 2004). Furthermore, the present results showed survey of biological and behavioral traits of a that P. persimilis would prey more on juvenile versatile predator. Redia 86:153-164. stages than on adult male or female stages of T. Eveleigh, E.S. & Chant, D.A. 1981. Experimental urticae under all situations when there were ample studies on acarine predator-prey interactions: effect preys. This was similar with T. bambusae Ehara of predator age and feeding history on prey fed with Schizotetranychus nanjingensis (Zhang et consumption and the functional response (Acarina: al. 1999). Again, while the food value of different Phytoseiidae). Can. J. Zool. 59 (7):1387-1406. prey stages are important, it appears that the Fernando, M.H.J.P. & Hassell, M.P. 1980. Predator– dietetic value of spider mite juvenile seems to prey responses in an acarine system. Res. Popul. equal to that of eggs. The present study showed Ecol. 22:301-322. that the P. persimilis could survive at low prey Friese, D.D. & Gilstrap F.E. 1982. Influence of prey density (about 10). Overall, the present study availability on reproduction and prey consumption of identified some characteristics listed by McMurtry Phytoseiulus persimilis, Amblyseius californicus and (1982) for a predatory mite to be considered a Metaseiulus occidentalis (Acarina: Phytoseiidae). successful biological control agent. The Int. J. Acarol. 8:85-89. performance of P. persimilis was greatly affected Fujii, K., Holling, C.S. & Mace, P.M. 1986. A simple by their food resources. generalized model of attack by predators and The predation model estimated in the present parasites. Ecol. Res. 1:141-156. study reveals that the expected number of prey Hassell, M.P. 1978. The Dynamics of Arthropod attacked similarly. This conclusion is supported by Predator–Prey System. (Monographs on Population the fractional values of the estimated χ2 goodness Biology, 13 Ed. May, R.M.) Princeton University of fit test, which are much lower than the table χ2 Press, Princeton, New Jersey, p. 237. value of 11.07 at P<0.05 with 5 degrees of Hassell, M.P., Lawton, J.H. & Beddington, J.R. 1977. freedom for both life stages and at different Sigmoid functional responses by invertebrate temperatures of this predator. This indicates the predators and parasitoids. J. Anim. Ecol. 46:249- best fit of the curves presented in Figs. 1-14. Our 262. results indicate that P. persimilis is highly effective Holling, C.S. 1959. Some characteristics of simple at higher temperatures by significantly increasing types of predation and parasitism. Can. Entomol. its attack rate as well as decreasing its prey 91(7):385-398. handling time. Although the effect of prey density is somewhat limited to generalize it for foraging Holling, C.S. 1961. Principles of insect predation. Ann. Rev. Entomol. 6:163-182. activity of P. persimilis in our data, it appears to perform well on leaves with some range of prey Holling, C.S. 1965. The functional response of density. P. persimilis can be a good biological predators to prey density and its role in mimicry and control agent of T. urticae under warm conditions. population regulation. Mem. Entomol. Soc. Can. 45:1-60. Acknowledgement: This is a part of the Ph. D. Huffaker, C.B. & Messenger, P.S. 1976. Theory and work of the first author and he acknowledges the practice of biological control. Academic Press, New fellowship given by the Institute of Biological York, 788pp. Sciences, University of Rajshahi and University Grants Commission, Bangladesh for this study. Huffaker, C.B, van deVrie, M. & McMurtry, J.A. 1970. Ecology of tetranychid mites and their natural enemies: a review. II. Tetranychid populations and their possible control by predators: an evaluation. Hilgardia 40:391-458. 8 Haque et al.
Hunter, A.F. & Elkinton, J.S. 1999. Interaction between Solomon, M.E. 1949. The natural control of animal phenology and density effects on mortality from populations. J. Anim. Ecol. 18:1-35. natural enemies. J. Anim. Ecol. 68:1093-1100. Takafuji, A. & Chant, D.A. 1976. Comparative studies of Hunter, M.D. & Price P.W. 1992. Playing chutes and two species of predaceous mites (Acarina: ladders: heterogeneity and the relative roles of Phytoseiidae), with special reference to their bottom-up and top-down forces in natural responses to the density of their prey. Res. Popul. communities. Ecology 73:724-732. Ecol. 17:255-310. Jolly, R.L. 2000. The predatory potential of Neoseiulus Taylor, R.J. 1984. Predation. Chapman & Hall. London, californicus: its potential as a biological control 166 pp. agent for the fruit tree spider mite. Panonychus ulmi. BCPC Conference, Brighton, Pest and Diseases J. Thongtab, T., Chandrapatya, A. & Baker, G.T. 2001. pp. 487-490. Biology and efficacy of the predatory mite, Amblyseius longispinossus (Evans)) as a biological McMurtry, J.A. 1982. The use of phytoseiids for control agent of Eotertranychus cendanai Rimando biological control: progress and future prospects. In: (Acarina: Tetranychidae). J. Appl. Entomol. M A Hoy (ed), Recent Advances in Knowledge of 125:543-549. the Phytoseiidae, pp. 23-48, Div. Agric. Sci. Univ. California Publ. 3284. Turchin, P. 1995. Population regulation: old arguments and a new synthesis. In: Cappuccino N and Price Mori, H. & Chant D.A 1966. The influence of prey PW (eds), Population dynamics, pp. 19-40, density, relative humidity, and starvation on the Acadamic Press, London. predaceous behavior of Phytoseiulus persimilis Athias-Henriot (Acarina: Phytoseiidae). Can. J. Zool. Zhang, Y.X., Zhang, Z.Q., Liu, Q. & Lin, J. 1999. 44:483-491. Biology of Typhlodromus bambusae (Acari, Phytoseiidae) a predator of Schizotertranychus Murdoch, W.W. & Oaten, A. 1975. Predation and nanjingensis (Acari: Tetranychidae). Syst. Appl. population stability. Adv. Ecol. Res. 9:1-131. Acarol. 4:57-62. Rasmy, A.H., Abdel-Rahman, H.A. & Hussein, H.E. 1991. Suitability of different mite prey for the development of the predatory mite, Phytoseiulus persimilis. Exp. Appl. Acarol. 11:89-91.
Manuscript received on 30.10.2010, Accepted on 07.02.2011 Univ. J. Zool. Rajshahi. Univ. Vol. 29, 2010 pp. 09-16 ISSN 1023-61041 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Seasonal abundance of zooplankton and growth performance of prawn and fish in ponds of Rajshahi MAB Siddique, M Afzal Hussain*, MA Hossain and M Manjurul Alam Department of Fisheries, University of Rajshahi, Rajshahi-6205, Bangladesh *Dr. M. Afzal Hussain, Associate Professor, Department of Fisheries, University of Rajshahi, Rajshahi-6205, Bangladesh.E-mail: [email protected] / Tel: 88-0721-750041-49 Ext. 4117(Off)/Fax: 88-0721-750064
Abstract: The present study was conducted on seasonal abundance of phytoplankton of prawn based carp polyculture system fishponds for a period of six months from September 2006 to February in the village Meherchandi of Boalia thana under Rajshahi district of Bangladesh. During the study period, it was found that water temperature varied from 16.59°C (T2) to 29.82 (T1), transparency 26.72 (T1) to 40.13cm (T2), pH 7.21 (T1) to 8.0 (T2) DO 2.13 (T2) to 3.6mg/l (T2), CO2 4.63 (T1) to 10.63mg/l (T2), NH3N 0.001 (T1) to 0.0197mg/l (T2) and total alkalinity fluctuated from 85.38 (T1) to 147.75mgl (T2). A total four groups of zooplankton were identified where 5 rotifera (38.41%) was dominant followed by 3 cladocera (31.49%), 2 copepoda (21.84%) and crustacean larvae (8.26%). It was also found that zooplankton showed abundance in February. The highest total average zooplankton (18390.79±2144.63 cells/l) was recorded in T1 than T2 which might be due to the effective uses by high stocking density of fishes. Zooplankton showed positive correlation with pH, DO, CO2 and alkalinity in T1. In case of T2, positive co-relationship was found with pH, DO, CO2 and NH3-N. The mean values of highest final weight of M. rosenbergii, H. molitrix and C. catla were 89.20±8.62g (T1), 682.70±44.20g (T2) and 428.00±4.50g (T1) respectively, highest survival rate was found 76.25±6.25% (T1) of prawn and highest total average yield was obtained 940.65±259.43 kg/ha in T2 due to high stocking densities of prawn. Key words: Zooplankton abundance, physico-chemical parameters, carp polyculture, growth performance
Introduction (1978), Bhuiyan & Nesa (1998a & 1998b), Islam et al. (2000), Chowdhury & Mamun (2006), The role of plankton in a water body of the Rahman & Hossain (2008) and many researchers sources of foods for fishes has been well worked on the percentage composition, seasonal recognized. Because plankton is the natural food variation and occurrence of freshwater of many species of fishes, especially zooplankton zooplankton. But, until now no systematic constitute important food items of many investigation was done on the zooplankton omnivorous and carnivorous fishes. The larvae of abundance of prawn based carp polyculture fish carps feed mostly on zooplankton (Dewan et al., pond in Rajshahi region. Therefore, the present 1977). According to Prasad & Singh (2003), the investigation has been undertaken on related to zooplankton forms the principal sources of food the title. for fish within the water body. Bhuiyan & Islam (1988) observed that zooplankton also plays a Materials and Methods very important role in the food chain as they are in the second trophic level as primary consumer and Location of study area and descriptions of also as contributors to the next trophic level. ponds Seasonal abundance of plankton in a fish pond The present study was conducted in the village are of great importance for any type of Meherchandi of Boalia Thana under Rajshahi aquaculture operation, as they vary from water district for a period of 6 months from September quality of culture ponds, location to location and 2006 to February 2007. For this study, four ponds within similar ecological conditions (Boyd, 1982). were selected under two treatments with an Among the different types of culture systems, the average area of 0.34 ha and depth of 1m. The semi-intensive aquaculture is common practice in ponds were rectangular in shape, well exposed to Bangladesh. That’s why a large number of sunlight, independent and completely free from research works has conducted on the ecology of aquatic vegetation. zooplankton population from different waters body of carp polyculture fish ponds such Khan et al. 10 Siddique et al.
Experimental design and pond preparation Study of growth performance The present experiment was conducted with two Final weight (g), survival rate (%) and yield (kg/ha) treatments each with two replications and every of prawn silver carp and catla was estimated. treatment was designed with M. rosenbergii, H. Survival rate and yield were estimated using the molitrix and C. catla. The stocking density of carp following formula in each treatment was fixed (H. molitrix-2968/ha C. catla-1976/ha) but the stocking density of fishNo.of harvested prawn was at the rate of 40/40m2 (9880/ha) in Survival rate (%) = ×100 2 fishofNo. stocked Treatment 1 (T1) and at the rate of 60/40m (14820/ha) in treatment 2 (T2). During the study period, all the ponds were limed at the dose of Yield/production = No. of fish harvested × final 250kg/ha after removal of aquatic weeds and weight of fish. unwanted species and fertilized with cowdung- Statistical analysis was done through SPSS 2470kg/ha, urea-50kg/ha and TSP-50kg/ha before programme and means were also compared to stocking. Shelter or refuges was made for prawn see the significant difference. by the uses of dried branches of bamboo which covered minimum 5% area of each pond. After Results and Discussion seven days of fertilization all the ponds were stocked with specific species and initial average Water quality parameters: During the study length, weights and sizes of each species of period some selected physico-chemical fishes and prawns were recorded before realizing. parameters were studied which are shown in Post stocking fertilization was made (cow dung- the Table 1. It was found that highest water 50kg/ha, urea-1.25kg/ha and TSP-1.25kg/ha) as temperature was found 29.82°C (September ’06) daily basis. The supplementary feeds were in T1 and lowest was 16.59°C (February ’07) in T2. applied twice daily for fish species with same Transparency was observed to be lowest feeding formulation (rice bran 20% + mustard oil (26.72cm) in February ’07 in T1 and highest cake 50% + fish meal 30%) at the rate of 5% of (40.13cm) in December ’06 in T2. The pond water fish. The daily ration was adjusted based on the of both treatments showed alkaline condition. The determination of total biomass through monthly highest pH was noted (8.0) in February ’07 in T2 sampling. and lowest was 7.21(October ’06) in T1. The Study of parameters lowest and highest dissolved oxygen content of water varied 2.13 (October’ 06) to 3.6mg/l The physico-chemical parameters were done (September’ 06) which was recorded in T2. Free fortnightly (8:30-9:30am). A centigrade thermometer carbon dioxide showed maximum (10.63mg/l) in used to recorded water temperature (°C). T2 in February’07 and minimum (4.63mg/l) in Measurement of water transparency (cm) was September ’06 in T1. NH3N varied from 0.001 done by a secchi disc of 15.5cm diameter. pH was (November’ 06 and February ’06) to 0.02mg/l measured by a pH indicator paper (LOGAK, (September ’06) in T1 and T2 respectively. The Korea). DO was determined by the Winkler’s total alkalinity fluctuated from 85.38 (September’ titration method (APHA, 1989). Free CO , NH -N 2 3 06) to 147.75mgl (September 06) where highest and total alkalinity was determined according to Welch (1948). and lowest was recorded in T1 and T2 respectively. During the study period it was also found that the Study of zooplankton mean values of different water quality parameters For zooplankton study, 20 liters of water sample were with the acceptable ranges (Table 1). Hoq et from each pond was collected (8:30-9:30am) in a al. (1996) and Ling (1969) reported that the water plastic bucket fortnightly and passed through temperature ranged from 27.5 to 30.5°C and 22 to plankton net of 55μ mesh size. Then the 32°C was suitable for the growth of prawn and the concentrated plankton samples were preserved in present finding almost similar with findings of plastic vials with 5% formalin for subsequent Hossain & Akhteruzzaman (2007). Secchi disc studies. For the qualitative and quantitative study of reading was lower in T1 and high in T2. However the plankton, 1ml of the concentrated samples was ranges of secchi disc reading were found favorable taken by a dropper and then put on the S-R for carp-prawn culture (Hossain et al., 2002) who (Sedgwick-Rafter) counting chamber as described recommended 35 to 60cm water transparency for by Stirling (1985). Zooplankton were identified up to prawn farming. pH values 6.5 to 9.0 (Swingle, 1967) genus level following keys were given by Ward and and 6.9 to 8.8 (Chiu, 1987) noted that optimum Whipple (1954), & Needham & Needham (1962). range for prawn culture. The best range of water pH for shrimp culture is 7-9 (Boyd & Faust, 1992). Therefore, the mean value of pH was found within 11
the suitable range according to the aforesaid NH3-N was in suitable range for fish culture and scientists. Comparatively lower value of DO might which is almost similar with findings of Boyd (1998) be associated to the decomposition of unused feed who suggested keeping the ammonia-nitrogen value and excreta of the fish (Boyd, 1998). The present lees than 0.1 mg/l. Singh & Singh (1975) concluded findings more or less similar with the findings of Hoq that alkalinity range from 30-125mg/l is favorable for et al. (1996) who reported DO ranged from 4.0 to 5.9 fish growth and the present finding is strongly mg/l for prawn farming and CO2 below 12 mg/l is agreed with the findings of New and Singholka suitable for fish culture (Boyd, 1998). The value of (1985).
Table 1. Monthly variation of water quality parameters of pond-1 and pond-2 (September, 2006 to February, 2007) Temperature Transparency DO CO NH -N Alkalinity pH 2 3 Months (°C) (cm) (mg/l) (mg/l) (mg/l) (mg/l) T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 Sep’06 29.82 29.59 32.63 38.94 7.41 7.38 3.10 3.6 4.63 6.00 0.004 0.019 85.38 147.75 Oct’06 28.16 29.07 31.5 32.55 7.21 7.53 2.79 2.13 8.38 6.63 0.012 0.018 94.13 104.25 Nov’06 24.21 24.10 34.69 38.32 7.57 7.59 3.38 2.96 6.38 7.13 0.001 0.019 97.38 102.75 Dec’06 18.23 18.59 35.63 40.13 7.49 7.64 3.26 2.99 7.63 7.64 0.04 0.019 93.25 113.88 Jan’07 18.88 19 27.82 33.32 7.67 7.63 2.56 2.93 9.38 9.50 0.011 0.019 92.75 110.75 Feb’08 16.75 16.59 26.72 27.68 7.89 8.00 2.98 2.84 9.63 10.63 0.001 0.015 122.38 123.13 Mean 22.68 22.82 31.50 35.16 7.54 7.63 3.04 2.91 7.75 7.92 0.012 0.018 97.55 117.09 ± SD ±5.53 ±5.61 ±3.60 ±4.80 ±0.23 ±0.21 ±0.29 ±0.47 ±1.95 ±178 ±0.01 ±0.00 ±12.79 ±16.72 *Acceptable 25-31 25-40 6.5-9 5-7 Below 12 Below 0.025 40-200 range
The occurrence (%) of different zooplankton (2184%, Cyclops>Diaptomus), and crustacean groups: The occurrences (%) of different groups larvae constitutes 8.26% of total zooplankton of zooplankton with genera under different groups. Rahman & Hossain (2008) study on four treatments are shown in Table 2 and Fig 1. groups of zooplankton of culture and non culture Dominance of different groups and genera were fishponds and recorded that 2 copepods identified on the basis of presence in both constituted 30.47 to 43.39%, 4 rotifers constituted treatments. During the present study, a total of 10 19.17 to 21.67%, 3 cladocera constituted 17.54 to genera under 3 groups of zooplankton were 27.09% and crustacean larvae constituted 15.28 identified where 5 rotifera (38.41%) was dominant to 19.91% in total zooplankton population. These group (Brachionus>Keratella>Notholca>Asplanchna findings are in good agreement with Hossain & >Filinia) followed by 3 cladocera (31.49%, Akhterruzzaman (2007). Daphnia >Moina> Diaphanosoma), 2 copepoda
Table 2. The occurrence (%) of different groups of phytoplankton with genera under different treatments during the study period Serial % of genus Zooplankton groups Genus No. T1 T2 Copepoda Cyclops 55.35 59.92 1 (21.84%) Diaptomus 44.65 40.08 Notholca 15.85 17.39 Keratella 24.18 19.22 Rotifera 2 Brachionus 40.28 37.4 (38.41%) Filinia 10.28 9.12 Asplanchna 9.41 16.87 Moina 34.77 38.46 Cladocera 3 Daphnia 44.85 41.33 (31.49%) Diaphanosoma 20.38 20.21 4 Nauplius (8.26%) 100.00 100.00
12 Siddique et al.
(2531.25 cells/l) was recorded in T2 (October, Crustacean Copepoda, 2006). The highest and lowest value of larvae, 8.26% 21.84% crustacean larvae varied from 3746.25 to 697.50 Cladocera, cells/l in T1 and T2 respectively where highest was 31.49% recorded in February, 2007 and lowest was recorded in September, 2006. During the study period it was also found that highest total average zooplankton (18390.79±2144.63 cells/l) was Rotifera, recorded in T1 than T2 which might be due to the 38.41% effective uses by high stocking density of fish (Table 3). Rahman & Hossain (2008) found peak season of copepods in September, rotifers Fig 1. The occurrence (%) of different groups cladocerans and crustacean larvae were zooplankton dominant in January. Das & Srivastava (1956) Seasonal abundance of zooplankton groups: divided their entire investigation year into various Monthly abundance of different zooplankton dominant periods such as September, October groups under different treatments are shown in and November constituted the copepodan period, Table 3 and Fig 2 and 3. . It was found that the December and January were characterized by highest concentration of copepoda was found cladocerans and April, May and June were 6851.25 cells/l (February, 2007) and lowest value characterized by crustacean larvae. From the was recorded 2542.50 cells/l (September, 2006) study, it was observed that zooplankton showed in T1. The concentration of rotifera varied from peak in January to February. Bhuiyan & Nessa 4050.00 to 11812.50 cells/l where highest was (1998a,b) and Islam et al. (2000) recorded highest recorded in T1 (February’07) and lowest was density of zooplankton in January (42213units/l recorded in T2 (October, 2006). The highest and 1350units/l respectively). The present finding concentration of cladocera (8268.75 cells/l) was is almost similar with the findings of Hossain & recorded in T1 (February, 2007) and lowest Akhterruzzaman (2007).
Table 3. Monthly abundance of different zooplankton groups under different treatments
Zooplankton concentration (cells/l) Groups Zooplankton groups Sep’06 Oct’06 Nov’06 Dec’06 Jan’07 Feb’07 total (cells/l) Treatment 1 Copepoda 2542.50 3208.75 3307.50 3780.00 5298.75 6851.25 Rotifera 4736.25 4387.50 5332.50 7728.75 8845.00 11812.5 18390.79 Cladocera 2995.00 2767.50 4185.00 6378.75 7240.00 8268.75 ±2144.63 Crustacean larvae 832.50 971.00 1113.75 2362.50 1552.50 3746.25 Total 11106.25 11334.75 13938.75 20250.00 22936.25 30678.75 Treatment 2 Copepoda 2812.50 3105.00 3780.00 4353.75 4387.50 5533.75 Rotifera 4183.75 4050.00 5737.50 8842.50 6506.25 9888.75 17195.04 Cladocera 2812.50 2531.25 3876.25 7089.00 6850.25 7188.75 ±1830.18 Crustacean larvae 697.50 945.00 843.75 3003.50 1856.25 2295.00 Total 10508.25 10631.25 14237.50 23288.75 19600.25 24906.25 13
7500 Correlation between water quality parameters T1 T2 6500 and zoooplankton
5500 It was found that zooplankton showed positive
4500 correlation with pH (r=0.859), DO (r= 0.352), CO2 (r=0.838) and alkalinity (r=0.656) where negative 3500 co-relationship was found with water temperature 2500 and transparency in T1 (Table 4). In case of T2, Concentration (cells/l) Concentration 1500 positive co-relationship was found with pH 500 (r=0.686) DO (r=0.045), CO2 (r=0.787) and NH3-N Crustacean Copepoda Cladocera Rotifera (r=0.248) where negative co-relationship was larvae found with other observed parameters. Rahman & Zooplankton groups Hossain (2008) found that zooplankton showed significant positive correlation with pH, DO and Fig 2. Mean concentration of different groups of CO2. Miah et al. (1981) and Alam et al. (1987), - zooplankton of T1 and T2 zooplankton showed positive correlation with CO3 - and HCO3 alkalinity. Patra & Azadi (1987) 35000 reported that inverse relationship existed between T1 T2 30000 zooplankton and water temperature which is strongly support the present study. 25000 20000 Growth performance of prawn and fish 15000 Final weight, survival rate and yield of different 10000 fish species under different treatments are shown
Concentration (cells/l) Concentration 5000 in Table 5. During the study period maximum final weight (g) of M. rosenbergii, H. molirix, and C. 0 Sep'06 Oct'06 Nov'06 Dec'06 Jan'07 Feb'07 catla was recorded as 89.20 (T1), 672.70 (T2) and Months 428.00 (T1) respectively. The present result is more or less similar with the findings of Sadek and
Fig 3. Monthly abundance of zooplankton of T1 and T2
Table 4. Relationship between water quality parameters and zooplankton under different treatments during the study period Zooplankton Water quality parameters T1 T2 Temperature (ºC) -0.905 -0.865 Transparency (cm) -0.536 -0.071 pH 0.859 0.686 DO (mg/l)-morning 0.352 0.045 Free CO2 (mg/l) 0.838 0.787 NH3-N (mg/l) -0.312 0.248 Alkalinity (mg/l) 0.656 -0.120
El-Gayar (1993), Islam et al. (1999) and Chand et maximum yield (kg/ha) was recorded as 851.53 al. (2002). The highest value of survival rate (%) (T2), 1496.60 (T1) and 542.53 (T2) where total was recorded as 76.25 (T1), 74.99 (T1) and 68.75 highest average yield (kg/ha) was recorded for (T2) for these species respectively, which was these species as 940.65 in T2 due to high stocking higher than the findings of Alam et al. (2001) who density of prawn. Chand et al. (2002) recorded the recorded 62.50% survival rate of M. rosenbergii yield 229 and 622 kg/ha for the M. rosenbergii and and 96.42% of H. molitrix. Chand et al. (2002) C. catla, respectively. In case of H. molitrix, Islam recorded 65-66% survival rate of C. catla. and the et al. (1999) recorded yield 927.15 kg/ha. 14 Siddique et al.
Table 5. Variation in the mean values of final weight, survival rate and yield of different fish species under different treatments Final weight (g) Survival rate (%) Yield (kg/ha) Level of Species T1 T2 T1 T2 T1 T2 significance 89.20 80.03 76.25 71.65 677.27 851.53 M. rosenbergii NS ±8.62 ±6.38 ±6.25 ±1.65 ±119.98 ±87.48 675.50 682.70 74.99 70.83 1496.60 1427.87 H. molitrix NS ±21.75 ±44.20 ±8.33 ±4.17 ±118.54 ±8.47 428.00 398.60 62.50 68.75 529.99 542.53 C. catla NS ±4.50 ±8.40 ±12.50 ±6.25 ±111.57 ±60.64 901.29 940.65 Total average yield NS ±300.68 ±259.43 NS=Non significant
References Alam, A.K.M.N., Islam, M.A., Mollah, M.F.A. & Haq, Chand, B.K., Patra, B.C. & Das, S.K. 2002. Growth and M.S. 1987. Status of zooplankton in newly production of freshwater prawn and carp under constructed ponds and their relations to some polyculture system. J. Interacademicia. 6(2):210- meteorological and limnological factors. 215. Bangladesh J. Fish. 10(1):83-88. Chiu, Y.N. 1987. Water quality management for Alam, M.J., Jahan, D.A., Pramanik, W.A. & Hoq, M.E. intensive prawn culture ponds. In: Edited by Y. N. 2001. Polyculture of freshwater prawn, Chiu, L. M. Santos & R. O. Juliano, V. P. Technical Macrobrachium rosenbergii (de man) with carps: Consideration for the Management and Operation effects of prawn stocking density. Bangladesh J. of Intensive Prawn Farms. Aquaculture Society, Fish. Res. 5(2):135-143. Iloilo, Philippines: pp.74-95. APHA, 1989. Standard methods for the examination of Chowdhury, A.H. & Mamun, A.A. 2006. Physico- water and waste water (4th ed.) American Public chemical conditions and plankton population of two Health Association, Washington. 1125pp. fishponds in Khulna. Univ. J. Zool. Rajshahi Univ. 25: 41-44. Bhuiyan, A.S. & Islam, M. 1988. Seasonal variation in the percentage composition of the food of Das, S.M. & Srivastava, V.K. 1956. Some new Xenontodon cancila. Univ. j. zool. Rajshahi Univ. observations on plankton from fresh water ponds 7:33-36. and tanks of Lucknow. Indian Sci. Cult. 21(8):466- 467. Bhuiyan, A. S. & Nessa, Q. 1998a. Seasonal variation in the occurrence of some zooplankton in a fish Dewan, S., Ali, M. & Islam, M.A. 1977. Study on the pond. Bangladesh J. Fish. Res. 2(2):201-203. size and pattern of feeding of fries and fingerlings of three major carps, eg. Labeo rohita (Ham), Catla Bhuiyan, A.S. & Nessa, Q. 1998b. A quantitative study catla and Cirrhina mrigala. Bangladesh J. Agri. of zooplankton in relation to the physico-chemical 2(2):223-228. conditions of a freshwater fishpond of Rajshahi. Univ. j. zool. Rajshahi Univ. 17:29-37. Hoq, M.E., Islam, M.M. & Hossain, M.M. 1996. Freshwater prawn (M. rossenbergii) with Chinese Boyd, C.E. 1982. Water Quality Management for pond and Indian carps in farmers’ pond. J. Aqua. fish culture. Elsevier Science Publishers B.V., 1000 Tropics. 11:135-141. Amsterdam, The Netherlands, 318 pp. Hossain, M.A. & Akhteruzzaman, M. 2007. Effect of Boyd, C.E. 1998. Water Quality for Aquaculture. stocking density on the growth performance of Research and Development series no. 43. Auburn prawn (Macrobrachium rosenbergii) and carp under University, Alabama, USA. 37p. polyculture system in northern Bangladesh. Int. J. Boyd, C.E. & Faust, A.W. 1992. Pond monitoring and Sustain. Agrid. Tech. 3(5):18-22. management. In: Faust A.W. & L.J. Lester (eds.). Hossain, M.A., Siddique, M.A.L. & Miaje, M.A.H. 2002. Marine Shrimp Culture: Principles and Practices. Development of low-cost feed for culture of giant Elsevier Science, Amsterdam. pp. 497-508. freshwater prawn (Macrobrachium rosenbergii) in ponds. Bangladesh J. Fish. Res. 4:127-134. 15
Islam, M.N., Khan, T.A. & Bhuiyan, A.S. 2000. Ecology Prasad, B.B. & Singh, R.B. 2003. Composition, and seasonal abundance of some zooplankton of a abundance and distribution of phytoplankton and pond in Rajshahi. Univ. j. zool. Rajshahi Univ. 19:25- zoobenthos in a tropical water body. 32. Nat.Envin.Pollut.Technol. 2:255-258. Islam, M.R., Saha, S.B., Mollah, M.F.A. & Rokeya, J.A. Rahman, S. & Hussain, M.A. 2008. A study on the 1999. Polyculture of Macrobrachium resenbergii abundance of zooplankton of a culture and a non- (de man) with carps in ponds. Bangladesh J. Fish. culture pond of the Rajshahi University campus. 22(1):39-44. Univ. j. zool. Rajshahi Univ. 27:35-41. Jhingran, V. G. 1988. Fish and fishes of India . Sadek, S.S. & El-Gayar, F.M.H. 1993. Production of the Hindustan Publishing Corporation (India), Delhi. giant freshwater prawn Macrobrachium rosenbergii reared in brackish water ponds (Egypt). Khan, Y.S.A., Salam, A.M.A. & Ahmed, M.K. 1978. Proceedings of the first international symposium on Cladocera of the River Buriganga, Dacca, aquaculture technology and investment Bangladesh, Bangladesh J. Zool. 6:73-83. opportunities. Ministry of Agriculture and Water, Ling, S. W. 1969. The general biology and development Riyadh, Saudi Arabia. pp. 296-303. of M. rosenbergii (de Man). FAO Fisheries Report. Singh, C.S. & Singh, K.P. 1975. Feeding experiments 3589-3619 pp. on Indian major carps in Terai ponds. J. Inland Fish Miah, M.I, Bhuiyan, N.I. & Dewan, S. 1981. A Soc. India. 7:212-215. comparative study of the rate of growth of major Stirling, H.P. 1985. Chemical and Biological methods of carps in relation to physico-chemical and biological rd water analysis for aquaculturists. Institute of factors. Proc.3 Nat. Zool. Conf. pp. 215-223. Aquaculture, University of Stirling, Scotland. pp Needham, J.G. & Needham, P.R. 1962. A guide to the 119. th study of freshwater biology. 5 ed. Liolden-day. Swingle, H.S. 1967. Relation of pH of pond water to Inc. San Francisco. p.106. shrimp suitability for fish culture. Proc. Pacific Sci. New, M. B. & Singholka, S. 1985. Freshwater prawn farming. Congres. 9(10):72-75. A manual for the culture of Macrobrachium rosenbergii. Ward, H.B. & Whiple, B.C. 1954. Freshwater Biology. FAO Fisheries Technical Paper (225). Rev. 1: 118 pp. 2nd ed. Jhon willey and Sons. Inc. New York. 1246 Patra, R.W.R. & Azadi, M.A. 1987. Ecological studies pp. on the planktonic organisms of the Halda river. Welch, P.S. 1948. Limnological methods, Mc. Graw-Hill Bangladesh J. Zool. 15(2):109-123. Book Company. Inc. New York. pp. 206-216.
Manuscript received on 30.10.2010, Accepted on 21.04.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 17-22 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Antibacterial and larvicidal potentials of Derris indica (Lamk.) Bennet. extractives Omar Ali Mondal1, M. Emdadul Haque2 and Nurul Islam3 1 Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh 2 Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh 3 Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh
Abstract: Chloroform extracts of the fruit shell, leaves, root bark, root wood, seeds, stem bark and stem wood of Derris indica were tested for their antibacterial and larvicidal potentials. Except the seed extract all other extracts offered activity against 15 pathogenic bacteria. The fruit shell extract showed activity against B. cereus, S.-β- haemolyticus and S. typhi; the leaf extract against Klebsiella sp. only; the root bark against B. cereus, B. megaterium, B. subtilis, S. -β- haemolyticus, S. typhi, S. dysenteriae and S. sonnei; the root wood extract against B. sereus, B. megaterium, B. subtilis, S. -β- haemolyticus, S. typhi, S. dysenteriae, S. shiga, S. sonnei, Klebsiella sp. and P. aeruginosa; the stem bark extract against B. cereus, B. subtilis, S. -β- haemolyticus and S. sonnei and the stem wood extract against B. cereus, B. megaterium, B. subtilis, S. -β- haemolyticus, S. typhi and S. dysenteriae. According to the intensity of activity against the selected bacteria the D. indica extracts could be arranged in a descending order of root wood> root bark> stem wood> stem bark> fruit shell> leaf extract. The minimum inhibitory concentrations (MICs) of the chloroform extract of root wood of D. indica were 128 μg/ml against S. -β- haemolyticus, B. megaterium and S. dysenteriae and 64 μg/ml against B. cereus; for the stem wood extract 128 μg/ml against S. -β- haemolyticus, B. megaterium, B. cereus and B. subtilis, and 64 μg/ml against S. dysenteriae. The root bark, root wood, seed and stem wood extracts showed efficacy against the 3rd instar larvae of Musca domestica with LC50 values in a descending order of root wood (3615.92 ppm) > seed (5538.07 ppm) > stem wood (12139.40 ppm) > root bark (16721.24 ppm). Key words: Chloroform extract, Derris indica, antibacterial and larvicidal activity.
Introduction rotenone has been extracted from the roots of th Derris, a genus of trees belonging to the family Derris sp. during first half of the 19 century. Fabaceae, inhabitants of India, SriLanka, The seeds are mainly valued for the oil obtained Bangladesh, Myanmer, Thailand, Malaysia, North from them, which has many industrial and Australia and Polynesia, occurs in the tidal medicinal uses. Powdered seed is valued as a forests, often along river and canal banks, febrifuge and tonic and used also in bronchitis and especially along the water-edges. It is a very well whooping cough, and the seed oil is used as a known medicinal plant. Alcoholic and aqueous soap liniment to treat scabies, herpes and extracts of the fresh bark and leaves of this plant rheumatism (Burkill, 1966). The stem bark is are reported to exhibit remarkable antibacterial fibrous and is used for cordage. It is also given activity against Micrococcus pyogenes var. aureus internally in bleeding piles (Haemorrhoids). A (Anon, 1969). The juice of the leaves is prescribed decoction of bark is used for beri-beri. However, in flatulence dyspepsia, diarrhoea and cough; it is few reports on the biological activity of this plant, also considered as a remedy for leprosy and the especially on insect larvae and pathogenic root juice is used in the treatment of gonorrhoea. bacteria are available, which led us to go through A hot infusion of the leaves is used as a this investigation. In addition an experimental medicated bath for relieving rheumatic pains, and endeavor had been attempted against the for cleaning foul ulcers and sores. The roots and housefly larvae, while the housefly transmit more seeds are known to be used as fish-poison by the than 20 human and animal diseases (Hicking, aborigins of Australia (Kirtikar and Basu, 1935), 1974) and pathogens that are harmful to livestock and a very well known plant derived toxin 18 Mondal et al.
(Cumming & Cooper, 2000); while bacterial Here, the serial tube dilution technique was pandemics are not unknown to mankind. Mondal followed using nutrient broth medium to determine and Islam (2008) found insecticidal activity of the the MIC values of chloroform extracts against the test plant while chloroform extracts of its different gram positive bacteria, Streptococcus-β- parts were screened against adult beetles of haemolyticus, Bacillus megaterium, Bacillus Callosobruchus maculates (F.). cereus and Bacillus subtilis, and gram negative pathogenic bacteria, Shigella dysenteriae. Materials and Methods Preparation of inoculum: Fresh cultures of the Preparation of plant materials for extraction: test organisms were grown at 37°C overnight on The fresh leaves, fruit shell, root bark, root wood, nutrient agar medium. Bacterial suspensions were seeds, stem bark, and stem wood of D. indica then prepared in sterile nutrient broth medium in were collected from the campus of the University such a manner that the suspension contained 107 of Rajshahi, Bangladesh. After drying under cells/ml, which was confirmed by O.D. (0.5) shade the plant materials were powdered in a measurement. grinder separately avoiding excess heat during grinding. Preparation of the sample solution: The root wood and the stem extracts were selected for the Chemical extraction of the collected materials: MIC test and were taken into different vials in a Chloroform was selected to extract seven different fixed amount (2.048 mg). The broth medium (2 parts of D. indica separately. The ground dried ml) was then added to each of the vials and materials were extracted with sufficient amount of agitated well to make sample solution whose chloroform (500g × 1500ml × 3 times) for each of concentration become 1024 μg/ml. The standard the test samples by the cool method just plunging antibiotic Ciprofloxacin solution (Reiner, 1980) the materials for 72 hours. Extracts, thus, was prepared through the same procedure to get obtained through filtration and evaporation of the solvent as residue kept in a refrigerator after the concentration 512 μg/ml. proper labeling. Procedure of serial tube dilution technique: Antibacterial screening: The agar diffusion i. Twelve (12) autoclaved test tubes were technique (Bauer et al., 1966; Barry et al., 1980; taken, nine of which were marked as 1, 2, 3, Vander & Vlietinck, 1991) was employed to 4, 5, 6, 7, 8, 9 and the rest were assigned as conduct antibacterial screening. Standard Cm = (medium), Cs = (medium + antibiotic discs of Ciprofloxacin (30 μg /disc) were Compound) and Ci = (Medium + Inoculum). used for comparison. Fifteen pathogenic bacteria ii. One ml of sterile nutrient broth medium was (six of which were gram-positive and the rest were added to each of the 12 test tubes. gram-negative) were selected for the test and were cultured at the Molecular Biology Laboratory, iii. One ml of sample solution was added to the Institute of Biological Sciences, Rajshahi first test tube and mixed well. University, Rajshahi. The test extracts were iv. One ml content from the first test tube was dissolved in respective solvents in such a manner transferred by the sterile pipette to the that the desired concentrations for application in second test tube and mixed uniformly. Then the disc have been obtained. 1 ml of this mixture was transferred to the Determination of Minimum Inhibitory third test tube. This process of serial dilution Concentrations (MICs): There are two methods was continued up to the ninth test tube. for the determination of the MIC: v. One drop (10 μl) of properly diluted inoculum a) Serial tube dilution technique or turbidimetric was added to each of the nine test tubes and assay, and mixed well. b) The paper disc plate technique or agar diffusion vi. For the control test tube, 1 ml of the sample assay. solution was added to the 1 ml nutrient broth, Antibacterial and larvicidal potentials of Derris indica 19
mixed well and a half of this mixture was in terms of mm. The results are shown in Table 1. discarded. This was to check the clarity of Among the 15 bacteria (6 gram-positive and 9 the medium in presence of diluted solution of gram-negative) only three (B. cereus, S. -β- the compound. haemolyticus and S. typhi) were responsive to the vii. 10 μl of the inoculum was added to the fruit shell extract giving promising inhibition zones, control test tube Ci to observe the growth of only Klebsiella sp. (gram-negative) was the organism in the medium used. responsive to the leaf extract giving inhibition viii. The control test tube Cm, containing the zone; B. cereus, B. megaterium, B. subtilis, S.-β- medium only, was used to confirm the haemolyticus, S. typhi, S. dysenteriae and S. sterility of the medium. sonnei were responsive to root bark extract giving ix. All the test tubes were incubated at 37.5°C promising inhibition zones; B. cereus, B. for 18-24 hours. megaterium, B. subtilis, S.-β- haemolyticus, S. Larvicidal screening: The food was prepared typhi, S. dysenteriae, S. shiga, S. sonnei, with 6.25 g of wheat bran and 0.5 g of milk Klebsiella sp. and P. aeruginosa to the root wood powder (Red Cow) and 12 ml of water as a total of extract giving promising inhibition zones; B. 19.5 g. For D. indica extracts 500 mg was cereus, B. subtilis, S.-β-haemolyticus and S. dissolved in 1 ml of the respective solvent sonnei were responsive to the stem bark extract (Chloroform) and mixed with the prepared food. giving promising inhibition zones; B. cereus, B. However, being volatile, the solvent was megaterium, B. subtilis, S. -β-haemolyticus, S. evaporated out shortly. To have a dose-effect to typhi and S. dysenteriae were responsive to the calculate toxicity by probit analysis 4 other stem wood extract giving promising inhibition successive doses were prepared and applied zones respectively at 200 μg/disc application, through the ½ serial dilution method. Thus the however it was only 07 mm at 50 μg/disc concentration of the extract in the food medium application for all the test organisms except was calculated as 12,820.5 ppm for the highest Klebsiella sp. which offered no clear zone, while dose (Dose A). However, depending on the all the inhibition zones of the test materials efficacy traced through pilot experiments doses mentioned above were compared to the inhibition made of D. indica root bark extract were zones given by the standard Ciprofloxacin 30 20,512.82, 15,384.60, 10,256.40, 5,128.20, μg/disc and results are mentioned in the Table 1. 2,664.10 and 1,282.05 ppm; for root wood extract It is clearly evident from the above investigations 15,384.60, 10,256.40, 5,128.20, 2,664.10, that the extracts of different parts of D. indica are 1,282.05 and 641.03 ppm; for seed extract the significantly active against most of the bacteria doses were 12,820.50, 6,410.25, 3,205.13, used in the screening. The larvicidal effect against 1,602.56, 801.28 and 400.64 ppm and for the the 3rd instar larvae of M. domestica was also stem wood extract 20,512.82, 15,384.60, found remarkable. Thus extensive studies are 10,256.40, 5,128.20, 2,664.10 and 1,282.05 ppm essential for the isolation of active compound(s) in V/V state. For each test 10 larvae were for the development of novel antibacterial, as well released in the treated food medium and 3 as insecticidal agents especially from the root replicates were set for each of the doses, while a wood and stem wood of this promising plant. control was also set for comparison. The data was read after 24 h of exposure. Results and Discussion Antibacterial effects: The anitbacterial activities of the test materials were determined by measuring the diameters of the zones of inhibition
20 Mondal et al.
Table 1. Antibacterial activity of the chloroform extracts of the different parts of D. indica in comparison with the standard Ciprofloxacin. Diameter of zone of inhibition (in mm) 50 and 200 µg/disc Ciprofloxacin 30 Test organisms Fruit shell Leaf Root bark Root wood Stem bark Stem wood µg/disc 50 200 50 200 50 200 50 200 50 200 50 200 Gram-positive S. aureus ------31 B. cereus 07 18 - - 07 18 07 19 07 17 07 21 30 B. megaterium - - - - 07 18 07 21 - - 07 21 28 B. subtilis - - - - - 15 - 23 - 15 07 21 33 S. lutea ------30 S. β .haemolyticus 07 19 - - 07 21 07 21 07 22 07 22 31 Gram-negative S. typhi - 17 - - 07 19 - 19 - - 07 20 35 S. dysenteriae - - - - - 18 07 19 - - 07 21 31 S. shiga ------07 19 - - - - 34 S. sonnei - - - - 07 19 07 20 - 17 - - 33 S. boydii ------34 E. coli ------33 Klebsiella sp. - - - 12 - - - 13 - - - - 31 P. aeruginosa ------17 - - - - 31 Proteus sp. ------30
MIC of D. indica extracts against the test bacteria: The extracts found promising during the antibacterial screening have been subjected to the MIC test, especially on the test bacteria the extracts showed activity. The results have been presented in Table 2 and Table 3.
Table 2. Minimum Inhibitory concentrations of the chloroform extract of D. indica root wood against pathogenic bacteria. Test tube No. 1 2 3 4 5 6 7 8 9 10 Cm Cs Ci Result Nutrient broth medium (ml) 1 1 1 1 1 1 1 1 1 1 1 1 1 of MIC Root wood extract (μg/ml) 512 256 128 64 32 16 8 4 2 1 0 512 0 (μg/ml) Inoculum added (μl) 10 10 10 10 10 10 10 10 10 10 0 0 10 S. β- haemolyticus - - - + + + + + + + - - + 128 B. Megaterium - - - + + + + + + + - - + 128 B. cerus - - - - + + + + + + - - + 64 S. dysenteriae - - - + + + + + + + - - + 128 Note: “+” = Growth “-” = No growth
Table 3. Minimum inhibitory concentrations of the chloroform extract of D. indica stem wood against pathogenic bacteria. Test tube No. 1 2 3 4 5 6 7 8 9 10 Cm Cs Ci Result Nutrient broth medium (ml) 1 1 1 1 1 1 1 1 1 1 1 1 1 of MIC Root wood extract (μg/ml) 512 256 128 64 32 16 8 4 2 1 0 512 0 (μg/ml) Inoculum added (μl) 10 10 10 10 10 10 10 10 10 10 0 0 10 S. β- haemolyticus - - - + + + + + + + - - + 128 B. Megaterium - - - + + + + + + + - - + 128 B. cerus - - - + + + + + + + - - + 128 B. subtilis - - - + + + + + + + - - + 128 S. dysenteriae - - - - + + + + + + - - + 64 Note: “+” = Growth “-” = No growth Antibacterial and larvicidal potentials of Derris indica 21
Larvicidal effects: Among the seven extracts the fruit shell, leaf and stem bark extracts didn’t show any activity, while the rest offered mortality to the 3rd instar larvae of M. domestica. The result is presented in Table 4 and illustrated in Fig. 1.
Table 4. Dose-mortality effect of D. indica extracts against M. domestica larvae. Time LC value 95% Confidence limits Test extract 50 Regression equation χ2 Value exposed (ppm) Lower limits Upper limits Root bark 24h 16721.24 9957.03 28080.65 Y=2.4438+1.178X 2.109 (4) Root wood 24h 3615.92 2394.88 5459.52 Y=1.1554+1.080X 1.923 (4) Seed 24h 5538.07 2485.48 12339.75 Y=2.5137+0.664X 0.842 (4) Stem wood 24h 12139.40 7763.46 18981.91 Y=0.2793+1.156X 1.017 (4)
Y = 0.022 + 1.178X Y = 1.155 + 1.080X
5.5 6
5 5.5
4.5 5
4 4.5 Empirical probit Empirical probit Empirical 3.5 4 33.544.5 2.5 3 3.5 4 4.5 Log dose Log dose
A B
Y = 0.279 + 1.1566X Y = 2.513 + 0.664X
5.5 5.5
5 5 4.5 4.5 4 Empirical probit Empirical Empirical probit Empirical 3.5 4 3 3.5 4 4.5 2.25 2.75 3.25 3.75 4.25 Log dose Log dose
C D
Fig. 1. Probit mortality lines of the chloroform extracts of A- root bark; B- root wood; C- stem wood and D- seeds of D. indica against M. domestica larvae. 22 Mondal et al.
It is clearly evident from the above investigations Barry, A.L. 1980. Procedure for testing antimicrobial that the extracts of different parts of D. indica are agents in agar media. In: Antibiotic in laboratory significantly active against most of the bacteria medicine (Lorian ed.). Willium Wilkins Co., used in the screening. The larvicidal effect against Bultimore. USA. pp. 1-23. rd the 3 instar larvae of M. domestica was also Bauer, A.W., Kirby, W.M.M., Sheries, J.C. & Turek, M. found remarkable. Thus extensive studies are 1966. Antibiotic susceptibility testing by a essential for the isolation of active compound(s) standardized single disc method. An. J. Clin for the development of novel antibacterial, as well Pathol. 45:493-496. as insecticidal agents, especially from the root Cumming, J.M. & Cooper, B.E. 2000. Arthropods wood and stem wood of this promising plant. associated with livestock dung: housefly, Musca domestica L. Animal protection systematic study Acknowledgement (APSS), ECORC, Canada, p. 2. The authors are very much thankful to the Hicking, N.F. 1974. Household insect pest. Associated University Grants Commission (UGC) and the Business Programme, London. p. 176. Ministry of Science and Information & Communication Technology (MOSICT) of Kirtikar, K.R. & Basu, B.D. 1935. Indian Medicinal Plants, Vol. III, 2nd ed. Basu, L. M., Allahadad, Bangladesh for financial support; and grateful to India. pp. 1593-2393. the Chairman, Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh and also Mondal, O.A. & Islam, N. 2008. Toxicity of chloroform to the Director of the Institute of Biological extracts of Derris indica Bennet. against Sciences, University of Rajshahi, Rajshahi-6205, Callosobruchus maculates (F.) adults. Univ. J. Zool. Rajshahi Univ. 27:95-96. Bangladesh for providing laboratory facilities. Reiner, R. 1980. Antibiotics: An introduction F. Hoffmann-La Roche and Company Ltd. References Switzerland, pp. 21-25. Anon, 1969. The Wealth of India - a dictionary of Indian Vander, B.D.A. & Vlietinck, A.J. 1991. Screening raw materials and industrial products. CSIR, New methods for antibacterial and antiviral agents from Delhi, 8:210. higher plants. In: Assay for bioactivity (K. Hostettmann, Ed). Academic Press, London, Burkill, J.H. 1966. A dictionary of economic products of 1:47-69. the Malay peninsula. Art Printing Works, Kuala Lumpur, Vol. 2.
Manuscript received on 21.12.2010, Accepted on 25.04.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 23-28 ISSN 1023-61041 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Efficacy of cypermethrin, deltamethrin and nimbicidine on fecundity and egg viability of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) R. Khatun, W. Islam and K.A.M.S.H. Mondal Institute of Biological Sciences, University of Rajshahi, Rajshahi, Bangladesh
Abstract: Fecundity and egg viability of Tribolium castaneum (Herbst) females were significantly (P<0.01) reduced by synthetic pyrethroids and an alkaloid containing insecticide nimbicidine. In control, the average number of eggs laid by each female/day was 12.18. The lowest number of eggs laid by each female/day was noted as 4.38 when combining nimbicidine with two pyrethroids i.e., cypermethrin and deltamethrin. The percentage of egg hatching without insecticide treatment was 95%. The lowest percent of egg hatching was 63.95 when the adult female was fed on flour medium treated with combined doses of cypermethrin, deltamethrin and nimbicidine. Among the treated insecticides, nimbicidine (80.69%) proved effective in reducing the fertility than those of cypermethrin (86.05%) and deltamethrin (88.52%).
Key words: Tribolium, cypermethrin, deltamethrin, nimbicidine, fecundity, fertility
Introduction Materials and methods Oviposition in Tribolium is influenced by different Fecundity environmental factors including moisture, T. castaneum adults were mass reared on fresh temperature (Park & Frank, 1948), relative flour medium in a glass jar kept in an incubator at humidity (Holdaway, 1932), flour medium 0 (Khalequzzaman et al., 1994) and conditioning of 30 C temperature without light and humidity the medium by the beetles living in it (Mondal & control. After pupation, the pupae were collected Port, 1985). A fertile female oviposits no eggs in by sieving the flour with a 500 micrometer sieve the first 3 days but after that at an increasing rate and sexed by microscopic examination of the upto 18 eggs per day (Ashford, 1970). exogenital process of the female pupae (Halstead, Oviposition in Tribolium is also reduced by 1963). The male and female pupae were kept in insecticides (Taher & Cutkomp, 1983, Rahman, the petri dish (9 cm diam.) and observed until the 1992; Kamaruzzaman, 2000; Hasnat, 2003), emergence of adults. quinones and botanicals (Rahman, 1992; Akhtar, 1997; Banu, 2004; Khanom, 2004). Ten days old adults of known sex were paired with one from each sex and twenty five such pairs Viability of eggs also depends on different were used for oviposition experiment. Each pair environmental factors such as temperature and relative humidity (Haque & Islam, 1978), food was kept in a glass vial (50×25 mm) containing 1g (Khan & Mazid, 1985; Khalequzzaman et al., of food either treated or untreated (acetone only). 1994) and conditioning of food (Mondal, 1987). The upper surface of vials were plugged with cotton wool. The vials were kept in an incubator at Some conventional insecticides (Taher & 0 Cutkomp, 1983; Amin, 2000; Hasnat, 2003), 30 C. Eggs were collected after every three days reduced the progeny production of Tribolium by sieving the flour media over a period of 45 species. days. The fresh food was supplied to each petri dish after every five days to avoid the conditioning Active ingredient, azadirachtin of neem plant exhibited profound effects on fertility of stored of given food by the beetle themselves (Mondal & product insect pests including Tribolium spp. Port, 1985). The adults used in the experiments (Khanam, 2003). were all survivors - those adults which were alive and perfectly normal in external appearance on However, there is meagre information on the effects of cypermethrin, deltamethrin and emergence from the pupae without any nimbicidine alone or in combinations on both deformities (Ashford, 1970). The flour media fecundity and progeny production of T. treated with cypermethrin (0.5 ppm), deltamethrin castaneum. So, present study has been (1ppm) and nimbicidine (6 ppm) individually and undertaken. their different combinations were also used in the 24 Khatun et al.
experiments. The same procedure was 1955; Taher & Cutkomp, 1983), pirimiphos-methyl maintained for each treatment. (Mondal & Port, 1985; Rahman, 1992; Kamaruzzaman, 2000), lindane (Taher & Egg viability Cutkomp, 1983) and deltamethrin (Hasnat, 2003). The eggs laid by the first 15 pairs of T. castaneum from each treatment were selected for the source Control (Untreat) Acetone rd th Cyper (0.05) Delta (1.0) of eggs. Eggs were collected from 3 to 45 days Nimb (6.0) Cyper+ Delta with equal interval of 3 days from introduction of Cyper+ Nimb Delta+ Nimb each pair to the experimental tubes/vials. The Cyper+ Delta+ Nimb collected eggs were then kept in separate petri 0 50 dishes for each treatment and incubated at 30 C 40 without light and humidity control. 30 20 Eggs were regularly observed with a microscope 10 0 and hatched larvae were carefully separated eggs laid of Number 6th 9th 3rd 21st using the method described by Mondal & 12th 15th 18th 24th 27th 30th 36th 39th 45th 33rd 42nd Parween (1997). The percent of viability was Days calculated on the basis of the total number of first instar larvae that hatched from the number of Fig 1. The average number of eggs laid by eggs used (Mondal, 1987). T. castaneum females at intervals of 3 days over a period of 45 days. Results and Discussion Fecundity The reduction of fecundity due to nimbicidine found in the present experiment is similar to the The results and statistical analyses are shown in findings of Banu (2004) who reported reduced Fig 1 and Table 1. The results were analyzed by fecundity in alkaloid, azadirachtin (technical grade Analysis of Variance and Duncan’s Multiple 98% a.i) treatments on both T. castaneum and T. Range Test (DMRT). confusum. The present result is also similar to Cypermethrin, deltamethrin and nimbicidine had those of some previous workers who reported the significant (P<0.001) effects on reducing the reduction in oviposition of Tribolium due to fecundity of T. castaneum in comparison to that of botanicals (Amin, 2000; Khanam, 2003; Khanom, the control. 2004). Azadirachtin incorporated into wheat flour reduced the fecundity significantly in both T. The reduction of laying eggs in both cypermethrin castaneum (Mukherjee & Ramachandran (1989) and deltamethrin treatments is similar to the and T. confusum (Hu & Chiu, 1993) results of previous workers who reported that reduced fecundity in Tribolium was due to the effects of sub-lethal doses of DDT (Loschiavo,
Table 1. The average number of eggs laid by T. castaneum females reared on fresh medium (control) and medium treated with cypermethrin, deltamethrin and nimbicidine alone and their different combinations. Eggs laid/day/female Treatment (ppm) Mean SE Minimum Maximum Control (untreated) 11.14+0.21a 9.38 12.98 Acetone (As solvent) 11.22+01a 10.24 12.27 Cypermethrin (0.5ppm) 8.55+0.09b 7.80 9.27 Deltamethrin (1.0ppm) 8.64+0.06b 7.91 9.02 Nimbicidine (6.0ppm) 8.24+0.05b 7.58 8.62 Cypermethrin (0.5)+Deltamethrin (1.0) 7.03+0.07c 6.62 7.73 Cypermethrin (0.5)+Nimbicidine (6.0) 5.52+0.07d 5.18 6.27 Deltamethrin (1.0)+Nimbicidine (6.0) 7.24+0.08c 6.47 7.71 Cypermethrin (0.5) + Deltamethrin (1.0) + 4.71+0.04d 4.33 5.02 Nimbicidine (6.0) Each experiment consists of 20 pairs (male: female = 1:1) and eggs collected at intervals of 3 days over a period of 45 days. Means followed by the same letters indicate no significant (P>0.05) difference in the number of eggs laid by female adults (Duncan, 1955). Efficacy of cypermethrin, deltamethrin and nimbicidine on T. castaneum 25
It is evident that in both insecticides (synthetic (P<0.001) reduced the fertility of eggs laid by T. pyrethroids) and nimbicidine (alkaloid) treatments, castaneum females in comparison to those of the female laid few eggs at the beginning of the control. oviposition period and the numbers generally The reduced rate of hatching in T. castaneum in increased in course of time. But the rate of the present experiment due to the insecticides - oviposition started to decrease after 30 days both cypermethrin and deltamethrin is similar to the in treatments and control (Fig 1). This might be due findings of Mondal (1987). Rahman (1992) and to prolonged period of feeding on treated medium Kamaruzzaman (2000) who reported significantly affecting the metabolism of adult beetles which (P<0.05) reduced fertility of Tribolium due to the ultimately reduced the fecundity (Taher & Cutkomp, effect of organophosphorus insecticide, pirimiphos- 1983). methyl. Taher & Cutkomp (1983) also observed Egg viability that both DDT and lindane significantly (P<0.05) reduced fertility of T. confusum in comparison to The results and statistical analyses are presented control. in Table 2. All the treatments significantly
Table 2. The numbers and percentage to egg hatching in T. castaneum reared on fresh medium (control) and medium treated with cypermethrin, deltamethrin and nimbicidine alone and their different combinations.
Egg Hatching Treatment (ppm) Total Eggs Used Total Eggs Hatched % Hatching + S.E Control (Untreated) 2704 2669 95.0+0.30a Acetone (Control as solvents) 2000 1721 94.75+0.34a Cypermethrin (0.5) 1960 1735 86.02+0.70b Deltamethrin (1.0) 1880 1517 88.52+0.56b Nimbicidine (6.0) 1485 1169 80.69+0.58b Cypermethrin (0.5) + deltamethrin (1.0) 1255 963 78.72+0.75c Cypermethrin (0.5) + Nimbicidine (6.0) 1645 1307 76.73+0.45c Deltamethrin (1.0) + Nimbicidine (6.0) 1082 692 79.73+0.55c Cypermethrin (0.5) + Deltamethrin (1.0) + 2573 2438 63.95+0.73d Nimbicidine (6.0) Each experiment consists of 20 pairs (male: female = 1:1) and eggs collected at intervals of 3 days over a period of 45 days. Figures followed by the same letters indicate no significant (P>0.05) difference in percentage of egg hatching (Duncan, 1955).
The reduced rate of egg hatching due to The present result confirms the assertion that the nimbicidine (azadirachtin) found in the present fertility of eggs in Tribolium depends on experiment is similar to the findings of Khanam & environmental factors including food media. In the Talukder (1993) who reported the reduced fertility present experiment, the fertility was found to in T. castaneum and T. confusum due to decline after 24 days exposure of T. castaneum to botanicals like Bishkatali (Polygonum hydropiper the treated media (Table 2). This might be due to L), Neem (A. indica), Nishinda (Vitex negundo L.) longer period of feeding on treated medium and Royna (Aphanamixis polystachya W. & A.). affecting the metabolism of adult beetles and Both Neem leaf and seed extracts were also creating bad odours which ultimately reduced the found to reduce the fertility of FSS-II and CTC-12 fertility (Taher & Cutkomp, 1983, Mondal, 1987). strains of T. castaneum significantly (P<0.05) in Depending on the stage of development oocytes comparison to that of control (Khanom, 2004). develop abnormally and resorbed vitellogenesis is Banu (2004) reported that azadirachtin declined inhibited. Eggs laid by females fed on food the fertility of both T. castaneum and T. confusum. medium treated with azadirachtin or neem The fertility of T. castaneum was minimized due to extracts often have poorly formed chronic Neem plant extract treatment (Mannan et al., surfaces and may be more sensitive to timgal 1993). attack and are often less fertile (Schmutterer, 1987). 26 Khatun et al.
According to (Loschiavo, 1955) chemical Holdaway, F.G. 1932. An experimental study of the treatments probably affect the physiological state growth of populations of the flour beetle Tribolium of the female which possibly induce a reduction in castaneum Herbst as affected by atmospheric both fecundity and fertility. The net reduction of moisture. Ecol. Monogr. 2: 261-304. both fecundity and viability of eggs clearly Hu, M.Y. & Chiu, S.F. 1993. Experiments on the demonstrates the potential means of reducing the effectiveness of some botanical insecticides in population of T. castaneum. controlling the confused flour beetle (Tribolium confusum). J. South-China Agric. Univ. 14: 4, 32-37. Acknowledgement Kamaruzzaman, A.H.M. 2000. Effects of cyromazine The authors are grateful to ACI Bangladesh for and pirimiphos-methyl on Tribolium castaenum supplying experimental samples of Cypermethrin, Herbst and Tribolium confusum Duval. Unpublished Deltamethrin and Nimbicidine and Institute of PhD Thesis, University of Rajshahi, Bangladesh. Biological Sciences, University of Rajshahi for 302 pp. laboratory facilities to conduct the experiment and Khalequzzaman, M., Khanam, L.A.M. & Talukdar, D. providing financial support. The first author is grateful 1994. Growth of Tribolium confusum Duval on to the Ministry of Education, Government of the wheat flour with various yeast levels. Int. Pest. Cont. People’s Republic of Bangladesh for granting 36: 128-130. deputation to pursue higher education. Khan, A.R. & Mazid, A. 1985. The oviposition and fertility of References the confused flour beetle, Tribolium confusum Duval (Coleoptera: Tenebrionidae) on barley and rice flour. Univ. Akhtar, N. 1997. Effectiveness of some plant oils J. Zool. Rajshahi Univ. 4:8-10. against Tribolium castaneum (Herbst) and Tribolium confusum Duval (Coleoptera: Tenebrionidae). Khanam, L.A.M. 2003. Toxicity of some indigenous Unpublished PhD Thesis, University of Rajshahi, plant materials against Tribolium spp. throughout Bangladesh. 307pp. ontogeny. Unpublished PhD Thesis, University of Rajshahi, Bangladesh. 300 pp. Amin, T. 2000. Synergistic action of some indigenous plant materials with insecticides against Tribolium Khanam, L.A.M. & Talukder, D. 1993. Effect of castaneum Herbst. Unpublished PhD Thesis, biskhatali Polygonum hydropiper L leaf and royna University of Rajshahi, Bangladesh. 255 pp. Aphanamixis polystachya Wall (Parker) seed coat extract on the fecundity and fertility of Tribolium Ashford, R.W. 1970. Some relationships between the confusum Duval (Coleoptera: Tenebrionidae). red flour beetle, Tribolium castaneum (Herbst) Bangladesh J. Sci. Ind. Res. 28(3):49-55. (Coleoptera: Tenebrionidae) and Lymphotropha trioli Ashford (Neogregarinidae: Schizocystidae). Khanom, N.P. 2004. Effect of neem plant extracts and Acta Protozool. 7:513-529. insect growth regulators on Tribolium castaneum (Herbst). Unpublished PhD Thesis, University of Banu, M.J.A. 2004. Effects of azadirachtin on Tribolium Rajshahi, Bangladesh. 339 pp. castaneum and Tribolium confusum. Unpublished PhD Thesis, University of Rajshahi, Bangladesh. Loschiavo, S.R. 1955. Rates of oviposition of Tribolium 324 pp. confusum surviving exposure to residues of pp DDT. Can. Entomol. 87:246-249. Duncan, D.B. 1955. Multiple range and multiple F tests. Biometrics 11:1-41. Mannan, A., Rahman, S.M. & Hossain, A. 1993. Reproductive potential of Tribolium castaneum Halstead, D.G.H. 1963. External sex differences in (Herbst) (Coleoptera: Tenebrionidae) feed on flour stored-products Coleoptera. Bull. Ent. Res. 54:119- treated with some plant extract. Tribolium Inf. Bull. 34. 33:90-94. Haque, I. & Islam, M.A. 1978. Effect of different food on Mondal, K.A.M.S.H. 1987. Effect of methylquinone, the longevity and fecundity of Coccinella repanda aggregation pheromone and pirimiphos-methyl on Thumb. (Coleoptera: Coccinellidae). Bangladesh J. fertility of Tribolium castaneum Herbst. Univ. J. Zool. Agric. Sci. 5(2):233-245. Rajshahi Univ. 26:21-26. Hasnat, H. 2003. Combined Action of Dimilin and Mondal, K.A.M.S.H. & Parween, S. 1997. Laboratory Deltamethrin on Tribolium castaneum (Herbst). culturing of flour beetles, Tribolium species. Unpublished Ph D Thesis, University of Rajshahi, Tribolium Inform. Bull. 37:153-162. Bangladesh. 273 pp. Efficacy of cypermethrin, deltamethrin and nimbicidine on T. castaneum 27
Mondal, K.A.M.S.H. & Port, G.R. 1985. Effect of Rahman, A.S.M.S. 1992. Combined action of methylquinone, aggregation pheromone and pirimiphos-methyl, synthetic methylquinone and pirimiphos-methyl on the fecundity of Tribolium botanicals on Tribolium confusum Duval. Unpublished castaneum Herbst (Coleoptera: Tenebrionidae). PhD Thesis, University of Rajshahi, Bangladesh. 232 Proc. Fifth Nat. Zool. Conf. Bangladesh. pp. 45-53. pp. Mukherjee, S.N. & Ramachandran, R. 1989. Effects of Schmutterer, H. 1987. Insect growth-disrupting and azadirachtin on the feeding, growth and fecundity-reducing ingredients from the neem and development of Tribolium castaneum Herbst. chinaberry trees. In CRC Handbook of Natural (Coleoptera: Tenebrionidae). J. Appl. Ent. Pesticides, Vol. III, Insect Growth Regulators, Part B 10(2):145-149. (Morgan ED and Mandara NB, eds). CRC Press, Boca Raton, Florida. pp. 119-170. Park, T. & Frank, M.B. 1948. The fecundity and development of the flour beetles, Tribolium Taher, M. & Cutkomp, L.K. 1983. Effects of sublethal confusum and Tribolium castaneum at three doses of DDT and three other insecticides on constant temperatures. Ecology 29:368-372. Tribolium confusum Duval (Coleoptera: Tenebrionidae). J. Stored Prod. Res. 19:43-50.
Manuscript received on 30.03.2010, Accepted on 01.01.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 29-32 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Effect of salinity on the survival and growth of mud crabling, Scylla serrata M.Y. MIA1, and M.M.R. Shah Bangladesh Fisheries Research Institute, Brackishwater Station, Paikgacha, Khulna-9280, Bangladesh 1 Department of Environmental Science & Resource Management, Mawlana Bhashani Science & Technology University, Santosh, Tangail-1902, Bangladesh
Abstract: An experiment was conducted for a period of eight weeks under laboratory conditions to determine the effect of different salinity levels on the survival and growth of crablings of mud crab, Scylla serrata. The different salinity levels tested were: 5 ppt, 10 ppt, 15 ppt, 20 ppt and 25 ppt. The differences in growth increment in terms of body weight (BW) were significant (p<0.05) among different salinity treatments. The specific growth rate (SGR%/day) values of 2.67, 2.85, 4.07, 4.39 and 4.46 at 5 ppt, 10 ppt, 15 ppt, 20 ppt and 25 ppt, respectively were found significant (p<0.05). The survival rates of crablings were found to be the highest (47.67%) at 25 ppt and lowest (10%) at 5 ppt (p<0.05). The results indicated that increase of salinity from 5 to 25 ppt had significant effect on the survival and growth of mud crablings.
Key words: Mud crabling, salinity, specific growth rate, survival rate
Introduction effect of salinity on the survival and growth of mud crablings of S. serrata. Salinity is an important environmental variable for estuarine organisms, as it has many important Materials and Methods physiological and ecological effects (Christiansen Salinity tolerance of mud crablings, S. serrata was & Costlow 1975, Anger 1991, Minagawa 1992). It studied for eight weeks under laboratory is also an important environmental factor for conditions at Brackishwater Station, Bangladesh estuarine crabs as it may affect growth through its Fisheries Research Institute, Piakgacha, Khulna. influence on food intake, conversion efficiency Crablings were collected from tidal water in the and activity, which are important components of Sankha River of Paikgacha region under Khulna the bioenergetic budget. It is commonly known district, Bangladesh. Salinity of the river water was that mud crab, Scylla serrata is a marine/estuarine about 15 ppt and collected crablings were species and can not survive without saline water. temporarily kept in same salinity for a day. Based on this, culture of this commercially important crab species has been undertaken in Different test saline waters (5, 10, 15, 20 and 25 saline water environment. Some studies have ppt) were prepared by mixing brine with tap water. been made on larval rearing and seed production The tap water was stored in plastic containers and of mud crab giving special emphasis on salinity aerated continuously at ambient room (Marichamy & Rajapackiam 1992, Zainoddin temperature (30°C). All salinities were checked 1992). Marichamy and Rajapackiam (1992) with a hand held refractometer to the nearest 1 studied on larval rearing and seed production of ppt. In total 15 fiber glass aquaria (50 L) were the mud crab, S. serrata with a salinity around 35 used for this experiment. Each aquarium was ppt. Zainoddin (1992), conducted a study on filled up with 30 liter saline water and few pieces rearing of the larvae of the mud crab, S. serrata in of gravel and stone for shelter of test specimens. Malaysia with a salinity ranging from 29 to 32 ppt. Several papers deal with the salinity tolerance, Ten crablings (carapace length: 1.43±0.21 cm; effect of salinities on larval development of carapace width: 2.06±0.29 cm and body weight: different species of crabs (Ong & Costlow 1970, 1.67±0.75 g) were released into each of five Mia & Shokita 1997, Hsueh 1998). As far as it is aquarium with five different salinity treatments known no studies have been made for the viz., T1 (5 ppt), T2 (10 ppt), T3 (15 ppt), T4 (20 optimization of salinity requirements of mud ppt) and T5 (25 ppt) and the experiment was crablings in Bangladesh. With this view, the replicated thrice. The crablings were fed daily with present study was conducted to evaluate the slaughterhouse waste @ 5% of the total biomass. 30 Mia & Shah
Tested saline water in each aquarium was Prinpanapong and Youngwanichsaed (1992) replenished (app. 60-80%) daily with new supplies cultured mud crabs separately in ponds for two of saline water. At the time of replenishment, months, where range of salinity during two months observations and counts were done for survival of period was 10-20 ppt (avg. 15 ppt) in pond 1 and crablings. Growth performance in respect of body 10-23 ppt (avg.16 ppt) in pond 2. The survival rate weight (BW) of crablings was recorded at weekly was 60.92% in pond 1 and 51.45% in pond 2. On interval. Growth gained by the crablings was the whole, the survival rate was 57.63%. From the expressed in Specific Growth Rate (SGR) by the above discussion, it is clear that mud crablings following formula: survive and grow within low salinity up to 23 ppt. Kathirvel (1980) reared juveniles (1.7-2.9 cm) for Specific Growth Rate (SGR%/day) = (log L – e 2 80 days at salinity ranges from 22-30 ppt and log L ) x 100/ (T – T ); where, L = Final weight of e 1 2 1 2 survival rates was 85%. In the present study, crabling (g), L = Initial weight of crabling (g), T – 1 2 highest survival of 47.67% with the highest salinity T = Duration of the experiment (day). 1 of 25 ppt indicates that higher salinity has a Results and Discussion positive influence on survival of mud crablings. Kathirvel (1980) also found that juveniles of S. The survival rates of mud crablings after eight serrata could survive at 4 ppt with acclimation. weeks with variations in different salinity levels are The lower survival rates (12.5-50%) were shown in Table 1. At the end of the experiment, recorded at 8 – 10 ppt without acclimation and at survival rates were 10, 13.33, 21.40, 39.83 and 51ppt with acclimation (12.5% survival) (Nair et al. 47.67 %, respectively in T1, T2, T3, T4 and T5. 1974). From their results and the results of the The highest survival of 47.67% was found in 25 present study indicating the euryhaline nature of ppt and the lowest of 10% in 5 ppt. Survival rate in mud crab species. 25 ppt (47.67%) and 20 ppt (39.83%) were significantly (p<0.05) higher than that of 5, 10 and The changes in body weight (BW) of mud 15 ppt; though no significant (p>0.05) difference crablings reared in five different salinities for eight on survival rate was found among 5, 10 and 15 weeks are shown in Fig. 1. Significant difference ppt. Survival rate of 39.83% and 46.67% (p<0.05) was observed from the changes of BW of respectively, in 20 and 25 ppt also showed no mud crablings during the experimental period significant (p>0.05) difference. High mortality was among the treatments which indicated that salinity observed with the continuation of the experiment. levels of 5 ppt to 25 ppt had an effect on the Mortality due to cannibalism was a common changes of their body weight. problem and this could have affected the results.
Generally, newly moulted crablings are attacked 18.00 by others which resulted in death. Mortality of T1 crablings due to cannibalism has been widely 16.00 T2 T3 reported (Iversen 1986, Ryther & Bardech 1974, 14.00 T4
Costlow 1967). Khan & Alam (1992) stated that ) 12.00 T5 mud crabs, S. serrata were distributed over a wide 10.00 range of salinity, from 2 ppt to oceanic waters, from the coast to the interior brackish waters and 8.00 are essentially euryhaline, but die beyond 70 ppt. Body (gm weight 6.00
Salinity tolerances and requirements for zoeal 4.00 stages and juvenile crabs are not same. Hill 2.00 (1974) reported that the first stage zoeae of S. 0.00 serrata are not tolerant to high temperature 12345678 (above 25°C) or low salinity (below 17.5 ppt). The Week salinity required for egg incubation was 32 ppt (Christopher 1992). It indicates that salinity Fig. 1. Effect of salinity on the growth of body requirements for crab’s survival and reproduction weight (g) of mud crablings, S. serrata. are not same. They survive and grow in lower salinity but are not able to reproduce. Effect of salinity on mud crabling 31
The specific growth rates (SGR%/day) are shown Hence, it could be concluded that mud crab is a in Table 2. SGR by weight recorded in mud euryhaline species but specific saline water is crablings were 2.67, 2.85, 4.07, 4.39 and 4.46 essential for its larval metamorphosis and %/day respectively, in 5, 10, 15, 20 and 25 ppt. breeding. Since the salinity of estuary and sea However, among the treatments significantly (p<0.05) highest SGR (4.46 %/day) was found in water is not fixed but related with some factors 25 ppt and the lowest (2.67 %/day) in 5 ppt. like temperature, current, turbidity etc, so further Present results indicate that higher salinity has a research is needed using more higher saline positive relation with growth. Experimental culture water above 25 ppt to at least 45 ppt. In the of mud crab, S. serrata was done for a growout present study, survival rate is lower than other period of 115 days by Samarasinghe et al. (1992) result (Kathirvel 1980) though it is similar with the where salinity varied from 22-32 ppt and SGR was results (12.5%) of Nair et al. (1974). recorded as 1.67%/day. In their study, SGR was small as adult crabs do not mould frequently like In the present study, cannibalism occurred within juvenile crabs. SGR was also recorded to be the crablings as newly moulted crablings came 3.33%/day by Mia et al (2003) in a seven weeks within the reach of other crabs. This happened laboratory trial using 25 ppt saline water. Their result is similar to the present study. From the due to the experiments being carried in a closed present results of SGR, it may be concluded that water body like fiber glass aquarium. This might higher salinity appears to be good for growth of be one of the factors affecting the lower survival of the mud crabling. mud crablings in this study.
Table 1. Mean survival of mud crablings, S. serrata in different weeks under different levels of salinities.
Survival (%) Treatment 1st week 2nd week 3rd week 4th week 5th week 6th week 7th week 8th week T1 (5‰) 98.33 90.00 67.67 43.33 30.00 23.33 16.67 10.00b T2 (10‰) 96.67 60.00 30.00 30.00 26.67 23.33 20.00 13.33b T3 (15‰) 100 93.33 86.67 73.33 56.67 30.00 26.66 21.40b T4 (20‰) 100 93.33 83.33 80.00 73.33 62.00 54.17 39.83a T5 (25‰) 100 96.67 93.33 88.33 80.00 65.00 61.67 47.67a *Different letters are significantly (p<0.05) different when compared with DMRT
Table 2. Specific growth rates of mud crablings, S. serrata reared in different levels of saline water.
Specific growth rates (SGR) Treatments Initial weight (g) Final weight (g) %/day
T1 (5 ppt) 1.95±0.07 8.75±1.50 2.67b T2 (10 ppt) 1.76±0.06 8.07±0.66 2.85b T3 (15 ppt) 1.13±0.52 11.09±1.24 4.07a T4 (20 ppt) 1.10±0.50 12.79±1.53 4.39a T5 (25 ppt) 1.18±0.19 14.41±0.88 4.46a *Different letters are significantly (p<0.05) different when compared with DMRT
32 Mia & Shah
References Possibilty of marine fish culture in the salt pan area at Tuticorin. Ind. J. Fish., 21(1):120-126. Anger, K. 1991. Effects of temperature and salinity on the larval development of the Chinese mitten crab Ong, K. S., & Costlow, J. D. JR. 1970. The effect of Eriocheir sinensis (Decapoda: Grapsidae). Marine salinity and temperature on the larval development Ecol. Prog. Ser. 72:103-110. of stone crab, Menippe mercenaria (Say), reared in the laboratory. Chesapeake Sci. 11:16-29. Christiansen, M.E. & Costlow, J.D. JR. 1975. The effect of salinity and cyclic temperature on larval Prinpanapong, S. & Youngwanichsaed, T. 1992. development of the mud crab Rhithropanopeus Rearing of mud crab (Scylla serrata). In: The Mud harrisii (Brachyura: Xanthidae) reared in the Crab. A report on the seminar convened in Surat laboratory. Marine Biol. 32:215-221. Thani, Thailand, pp. 191-194. Christopher, L. 1992. A brief overview of the ecology Ryther, J. & Bardech, J. 1968. The status and potential and fisheries of the mud crab, Scylla serrata, in of aquaculture, particularly invertebrate and algae Queensland. In: The Mud Crab. A report on the culture. U.S. Dept. Comm. pp. 177-767, p. 261. seminar convened in Surat Thani, Thailand, pp. 65- Samarasinghe, R.P., Fernando, D.Y. & de Silva, O.S. 70. S.C. 1992. Pond culture of mud crab in Srilanka. In: Costlow, J.D. 1967. The effect of salinity and The mud crab, C. A. Angel (ed.). pp. 161-164. temperature on survival and metamorphosis of Proceeding of the seminar on mud crab in Surat megalops of blue crabs, Callinectes sapidus. Thani, Thailand, 5-8 November, 1991. Bay of Helgolander. Wiss. Meereunters. 15:84-97. Bengal, Madras. Hill, B.J. 1974. Salinity and temperature tolerance of Zainoddin, B.J. 1992. Preliminary studies on rearing the zoea of the portunid crab, Scylla serrata. Marine larvae of the mud crab (Scylla serrata) in Malaysia. Biol. 25(1):21-24. In: The Mud Crab. A report on the seminar convened in Surat Thani, Thailand, pp. 143-147. Hsueh, P.W. 1998. Salinity tolerance of hard stage Pinnotheres bidentatus (Decapoda: Brachyura: Pinnotheridae). Crustacean Res. 27:82-87. Iversen, E.S. 1986. Farming in the edge of the sea. Fishing News Books, UK. 301 pp. Kathirvel, M. 1980. Abundance of portunid crab seeds in Cochin backwater. In: Proc. Symp. On Coastal Aqua, Mar. Biol. Assn. Ind., Abs. No. 94. Khan, M.G. & Alam, M.F. 1992. The Mud Crab (Scylla serrata) fishery and its Bio-Economics in Bangladesh. In: The Mud Crab. A report on the seminar convened in Surat Thani, Thailand, pp. 29- 40. Marichamy, R. & Rajapackiam, S. 1992. Experiments on larval rearing and seed production of the mud crab, Scylla serrata. In: The Mud Crab. A report on the seminar convened in Surat Thani, Thailand, pp. 135-141. Mia, M.Y. & Shokita, S., 1997. Optimal salinity required for the larval development of two grapsid crabs, Helice leachi Hess and H. formosensis Rathbun. Crustacean Res. 26:70-74. Mia, M.Y., Rheman, S. & Alam, M.J. 2003. Comparative study of two feeds of mud crab, Scylla serrata. University j. zool. Rajshahi Univ. 22:125-130 Minagawa, M. 1992. Effects of salinity on survival, feeding and development of larvae of the red frog crab Ranina ranina. Nippon Suisan Gakkaishi. 58(10):1855-1860. Nair. R.V., Bensam, P. & Marichamy, R. 1974.
Manuscript received on 20.12.2010, Accepted on 07.02.2011 ISSN 1023-6104 DryUniv. and j. zool. wet Rajshahi. season Univ.polymorphism Vol. 29, 2010 in thepp. butterflies33-40 1 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Dry and wet season polymorphism in the butterflies, Melanitis leda and Mycalesis mineus (Satyridae: Lepidoptera) A.T.M.F. Islam *1, M.A. Razzak1, M.H. Islam1, A.S.M. Saifullah1, K. Endo2 and Akira Yamanaka2 1Institute of Food & Radiation Biology, Atomic Energy Research Establishment, G..P.O.Box-3787, Dhaka-1000, Bangladesh 2Department of Physics, Biology & Informatics, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan
Abstract: A field survey of butterflies was carried out in different areas of deciduous forests at Kaliakyar, Gazipur, during January-December, 2008. Seven species of satyrid butterflies were collected and two species, Melanitis leda and Mycalesis mineus were found to exhibit dry and wet season polymorphism. The dry season forms (DSFs) and wet season forms (WSFs) of the both species were distinguished by their upperside and underside wing patterns. The DSFs occurred only in the drier period (November to February) when day length was shorter than 12 hrs. at lower temperatures (24.1-20.6°C). Both the species of butterflies of the wetter period (March - October) were predominant of the WSF when day length was longer than 12 hrs. at higher temperatures (26.8-27.6°C). The fluctuations in the number of seasonal forms of M. leda and M. mineus were higher in DSF than those of WSF. The transition of DSF to WSF and WSF to DSF of M. leda was found to occur around the end of April and the early of September, respectively. But the turnover of DSF to WSF occurred around the early March and the turnover of WSF to DSF in October in M. mineus.
Key word: Butterflies, seasons, polymorphism, Melanitis leda and Mycalesis mineus
Introduction By contrast, wet season forms (WSF) are reproductively active, generally do not aggregate Many animals became extinct because they were in refugia, and are characterized by conspicuous unable to adapt to changes in their environment. eye-spot patterns on the under side of the wing However, many organisms remain alive under (Corbet & Pendlebury 1978, Kirk 1982, Brakefield cyclic (daily, monthly and seasonal) changes of & Larsen 1984, Brakefield & Reitsma 1991, Braby, environmental conditions to which they show 1994). The incidence of seasonal polymorphism in adaptation by decreased physiological activities or tropical environments has received little attention by changing their morphology. In tropical and (Shapiro 1976, Tauber et al. 1986). But there is no sub-tropical environments with contrasting dry and report on the seasonal polymorphism (DSF and wet seasons, many insects move into and WSF) of Melanitis leda and Mycalesis mineus in aggregate in moist refugia during the long and Bangladesh. This paper addresses their seasonal unfavourable dry season (Denlinger 1986, Jones changes in the wing phenotype, and fluctuation in 1987, Scheermeyer 1993). An alternative strategy the number (WSF and DSF) of both species. It may occur in some butterflies which become more also suggests possible factors inducing phenotypic cryptic during the dry season (Brakefield & Larson change of DSF and WSF in M. leda and M. 1984, Brakefield & Reitsma 1991, Windig 1992, mineus. Jones 1992). Materials and Methods Several tropical satyrid butterflies show striking seasonal polymorphism in the underside wing Butterflies: The butterflies (Melanitis leda and patterns. The dry season forms (DSF) are usually Mycalesis mineus) were collected from five reproductively inactive or less active and different areas in the deciduous forests at long-lived, aggregate in moist refugia and are Kaliakyar, Gazipur. Field surveys and collections characterized by reduced or no ocelli (eye-spots). of butterflies were carried out throughout the year 34 Islam et al.
(January to December) in 2008. The 5 research absence of eye-spots, colour and band pattern). sites were visited on sunny days of every month The body portions of butterflies were represented (2008) and butterflies were collected by using by terms used by Islam et al. 2007. insect nets. Collected butterflies were immediately Photography: Photographs of butterflies (dorsal killed by pressing the thorax carefully for and ventral sides) were taken by a digital camera minimizing damages to their external organs (Nikon, COOLPIX 5700, 5.0 mega pixels), (scales, legs and wing veins), put in triangular uploaded into a personal computer and processed envelopes of papers and brought to the laboratory. by using imaging software of Adobe Photoshop Date, time and place of collection site were LE5. Variations of the wing patterns of DSF and recorded for each specimen. Meteorological data WSF adults of M. leda and M. mineus. of each collection day were recorded and summarized in Table 1. Results and Observation Seasonal forms identification: Species of M. leda and M. mineus adults were found in two butterflies were identified according to the distinct seasonal morphs, viz. dry season form Specimen Books of Butterflies (Islam et al. 2007; (DSF) and wet season form (WSF). DSF was Pinratana, 1988). Polymorphic forms (WSF and found during March to October when day length DSF) were identified according to the was longer with high temperature whereas WSF characteristics of their wings, such as shapes was found during November to February when day (angular/regular) and patterns (presence or length was shorter with low temperature.
Table 1. Monthly temperature, humidity and rainfall data of the butterflies sampling areas in 2008 Month Temperature (ºC) Average Relative Total Rainfall Maxi Min Average Humidity (%) (mm) January 24.4 14.5 19.4 49.9 23 February 26.1 15.2 20.6 36.3 56 March 31.7 23.0 26.8 45.1 45 April 34.5 24.5 29.5 42.9 91 May 34.7 24.9 29.8 52.7 205 June 31.5 26.5 29.0 66.5 577 July 31.7 26.4 29.1 71.8 365 August 32.1 27.3 29.7 69.5 319 September 32.5 26.2 29.4 65.3 279 October 31.4 23.8 27.6 59.2 227 November 29.7 18.4 24.1 40.6 0 December 25.6 17.0 21.2 54.4 0 Mean 30.5 22.3 26.4 54.5 182.3
M. leda: tail on the hind wing more pronounced. Sub-marginal dark white-centered fulvous-ringed Dry Season Forms (DSFs): The DSF of M. leda ocellus (eye-spot) in area 2 absent in the hind wing has much smaller ocelli (eye-spots), longer tails, (Plate 1). larger wings and a variable background. Underside: Eye-spots reduced to minute white Upperside: Ground colour brown, the markings, dots and the regular irroration of the WSF replaced especially the ferruginous lunules inwardly by a highly variable cryptic pattern ranging in bordering the black sub-apical spots on the fore ground colour from a light tan over darker brown wing, larger, more extended below and above the and chestnut to almost black. Posterior wing black costa. Fore wing strongly angled near the without an eye-spot in area 2, but posteriorly apex. Eye-spots of the fore wing are more replaced by three or four minute white prominent and surrounded by orange line. Small sub-marginal spots (Plate 1).
Dry and wet season polymorphism in the butterflies 35
Plate 1. Upperside (a) and Underside (b) views of Melanitis leda DSF adult
Wet Season Forms (WSFs): The wet season fulvous-ringed eye-spot sub-terminally in forms of M. leda are with large, well differentiated interspace 2 and the apical eye-spot, sometime eye-spots, short tails, smaller wings. also other of the eye-spots, on the underside showing through (Plate 2). Upperside: Ground colour similar to that in the DSF. Apex of fore wing termen sub-acute, termen Underside: Underside paler and densely striated slightly angulated just below apex, or straight. Fore with dark brown. Fore wing with a discal curved wing with two large sub-apical black spots, each dark brown, narrow band, a post-discal similar with a smaller spot outwardly of pure white oblique band, followed by a series of four inwardly bordered, the inner edge of this black eye-spots that in interspace 8 the largest. Hind patch bordered narrowly with ochraceous, wing with a series of six eye-spots, the apical and interrupted in area 4, costal margin narrowly pale. sub-tornal the largest (Plate 2). Hind wing with a dark, white-centered
Plate 2. Upperside (a) and Underside (b) views of Melanitis leda WSF adult. 36 Islam et al.
Seasonal fluctuations of the numbers of WSF M. mineus: and DSF adults of M. leda Dry Season Forms (DSF): WSF and DSF adults appeared in April and Upperside: Ground colour was dark brown but September, and formed a peak in May and pale area of the fore wing often very extensive. November, respectively. The peak of the DSF was Fore wings have a large eye-spot with more than 100 times larger as compared to that of white-centre in space 5. Ventral portion of the hind the WSF. The transitions of DSF to WSF and WSF wing bears a small black spot (Plate 4). to DSF were found to occur around the end of April and the early of September, respectively (Fig. 3). Underside: Underside of the DSF deep chocolate brown to dusky brown of a darker shade. Basal half of the wings conspicuously darker than outer portion, the whole surface irrorated with fine brown striae, sometimes and a distinct dark discal band cross both wings. Eye-spots reduced to minute white dots, the posterior four on the hind wing in a straight line as in the WSF (Plate 4). Wet Season Forms (WSFs): Upperside: Ground colour dark brown. Fore and hind wings with slender subterminai and terminal pale lines. Fore wing with a single white-centered, fulvous or dark yellow-ringed, black eye-spot, Fig. 3. Seasonal fluctuations in the number of DSF generally set in a square pale area, in interspace 2, and WSF of M. leda adults (○ and ●) and the occasionally a similar smaller eye-spot without any average temperature (a broken line) pale surroundings area in interspace 5. Hind wing uniform, sometimes with one or two obscure post-median eye-spot (Plate 5).
Plate 4. Upperside (a) and Underside (b) views of Mycalesis mineus DSF adult
Underside: Ground colour similar to the upperside. they are deeply indented in space 4 and 5. Fore In the fore wing, there are two eye-spots situated and hind wings crossed by a transverse only in space 2 and 5. In the hind wing, there is dusky-white discal band, well-defined inwardly, line of eye-spots inwardly bordering by a outwardly diffused, followed by a post-discal series dusky-yellowish, sometimes purplish-white and of eye-spots (Plate 5). Dry and wet season polymorphism in the butterflies 37
Plate 5. Upperside (a) and Underside (b) views of Mycalesis mineus WSF adult Seasonal fluctuations in the numbers of WSF Larsen (1984) described the occurrence of dry and and DSF adults wet season forms in a variety of tropical satyrid butterflies. The WSF and DSF are well separated WSF and DSF adults appeared in April and by the number, shape, size, position and colour October, and formed a peak in May and November, composition of eye-spots, tail and wings and the respectively. The peak of the DSF was far higher background colour pattern. The WSFs in M. leda as compared to that of the WSF. The transitions of and M. mineus are characterized by a well DSF to WSF and WSF to DSF were found to occur developed sub-marginal ring of eye-spots on the around the early of March and October, brown ventral surface of the wings while in the respectively (Plate 6). DSF these are greatly reduced or absent. The development of seasonal morphs is determined by one or more environmental cues changing seasonally. The environmental control of the seasonal morphs is mediated by the hormonal system (Endo & Funatsu, 1985; Endo & Kamata, 1985). The induction of seasonal morphs development in tropical butterflies have been partly established for only a few species (McLeod 1968, 1984; Jones et al., 1985; Rienks 1985; Brakefield & Reitsma 1991; Jones 1992; Windig 1992). Janzen (1984) reported that in several Lepidoptera, temperature is of primary importance in regulating wing phenotype. Temperature was Fig. 6. Seasonal fluctuations in the number of DSF found to interact with photoperiod in pierid and WSF M. mineus adults (○ and ●) and the butterflies in the north of Australia and average temperature (a broken line) experiencing a small annual change in day length (Rienks, 1985). MCLeod (1968 & 1984) concluded that temperature was the only factor involved in Discussion the control of adult polymorphism in the nymphalid Seasonal polymorphism in the tropics and Precis octava in east Africa. Brakefield & Reitsma sub-tropics is an important ecological alternative to (1991) and Windig (1992) showed that different migration in space or time (diapause). Brakefield & temperatures in final instar larvae of Bicyclus safitza induced different seasonal forms. Jones 38 Islam et al.
(1992) also observed that the temperature strongly References: influenced polyphenism in Eurema spp., although temperature alone could not account for all the Braby, M.F. 1994. Effect of temperature and hostplants variations produced. Rienks (1985) and Jones et on survival, development and body size in three al. (1985) have also demonstrated that small tropical satyrine butterflies. Aus. J. Zool. changes in photoperiod in conjunction with 42:195-213. temperature can affect wing phenotype in two Brakefield, P. M. 1987. Field studies of seasonal tropical pierids. Brakefield & Larsen (1984), polyphenism in the butterfly Melanitis leda. Bull.. Brakefield (1987) and Brakefield & Reitsma (1991) British Ecol. Soc. 18:23-25. proposed that the alternative phenotypes (i.e. WSF and DSF) represent responses to seasonal Brakefield, P.M. & Larsen, T.B. 1984. The evolutionary differences in environmental conditions and significance of dry and wet season forms in some selective pressures such as the nature of tropical butterflies. Biol. J. Linn. Soc. 22:1-12. predation. The prominent eye-spot patterns of WSF is thought to rely on anti predator devices Brakefield, P.M. & Reitsma, N. 1991. Phenotypic which are displayed at rest and function principally plasticity, seasonal climate and the population in the deflection of attacks from vertebrate biology of Bicyclus butterflies (Satyridae) in Malawi. predators like lizards. In contrast, DSF butterflies Ecol. Ent. 16:291-303. have reduced or lacking of eye-spots and probably Corbet, A.S. & Pendlebury, H.M. 1978. The butterflies relies wholly on crypsis for survival. on the Malay Peninsula, 3rd edition, revised by J.N. In tropical and sub-tropical areas near the equator, Eliot. Malaysian Nature Society: Kuala Lumpur. photoperiod, the length of which shows a little Denlinger, D.L.1986. Dormancy in tropical insects. A. fluctuation, may not be a suitable indicator for Rev. Ent. 31:239-264. butterflies to determine the seasonal morphs development, such as WSF and DSF. In Satyrinae, Endo, K. & Funatsu, S. 1985. Hormonal control of the environmental factors regulating the seasonal seasonal morph determination in the swallowtail morph development (WSF and DSF) were not butterfly, Papilio xuthus L. (Lepidoptera: apparent yet. However, we have an interesting Papilionidae). J. Insect Physiol. 31:669-674. report that WSF adult of M. leda were developed in the wet season by subjecting larvae to a relatively Endo, K. & Kamata, Y. 1985. Hormonal control of low humid (60%) condition (Owen, 1971). Roskam seasonal-morph determination in the small copper & Brakefield (1999) indicated that M. leda adults butterfly, Lycaena phlaeas daimio Seitz. J. Insect having the wings of DSF are developed by Physiol. 31:701-706. subjecting larvae to a low temperature Islam, A.T.M.F., Razzak, M.A., Mamun, A.N.K., experienced in the rainy season. Saifullah, A.S.M., Shahjahan, R.M., Endo, K., The seasonal fluctuation in the number of M. leda Yamanaka, A., Kometani, M. & Inoue, M. 2007. and M. mineus were more or less similar in the Common Butterflies in Bangladesh. IFRB, Atomic series of above results. The turnover from DSF to Energy Research Establishment, Ganakbari Savar, WSF coincided with rising temperature when Dhaka. p. 33, with 16 plates. increase in humidity and food plants occur. In contrast, the turnover from WSF to DSF was found Janzen, D.H. 1984. Weather-related color at declining temperature, resulting in low humidity polymorphism of Rothschildia lebeau (Saturniidae). and less food plant Brakefield (1987) and Bull. Entomol. Soc. Amer. 30:16-20. Brakefield & Reitsma (1991) reported similar Jones, R.E.1987. Reproductive strategies for the observation. Seasonal polymorphism is linked with seasonal tropics. Insect Sci. Appl. 8:515-521. seasonal changes in breeding status which in turn is associated with changes in habitat favourably. Jones, R.E. 1992. Phenotypic variation in Australian Eurema species. Aust. J. Zool. 40:371-383. The results of our study imply that temperature, photoperiod and humidity play a crucial role in Jones, R.E., Rienks, J. & Wilson, L. 1985. Seasonally regulating seasonal morphs development in and environmentally induced polyphenism in M. leda and M. mineus. Eurema laeta lineata (Lepidoptera: Pieridae). J. Aust. Entomol. Soc. 24:161-167. Dry and wet season polymorphism in the butterflies 39
Kirk, W.D. 1982. Variation of fore wing spot number in Roskam, J.C. & Brakefield, P.M. 1999. Seasonal the mineus group of Mycalesis (Lepidoptera, polyphenism in Bicyclus (Lepidoptera: Satyridae) Satyridae) in S. E. Asia. Malay. Nat. J. butterflies: different climates need different cues. 35:229-236. Biol. J. Linn. Soc. 66:345-356. McLeod, L. 1968. Controlled environment experiments Scheermeyer, E. 1993. Overwintering of three with Precis octavia Cram. (Nymphalidae). J. Res. Australian danaines: Tirumala hamata hamata, Lepid. 7:1-18. Euploea tulliolus tulliolus and E. core corrinu. In Malcoim, S.B. and Zalucki, M.P. (eds.). Biology McLeod, L. 1984. Seasonal polyphenism in African and conservation of the monarch butterrfly: pp. Precis butterflies. In: R. I. Vane-Wright & P. R. 345-353. No. 38 Science Series, Natural History Ackery (Eds.), The Biology of Butterflies, Museum of Los Angeles County. Symposium of the Royal Entomological Society of London, 11:313-315. Shapiro, A.M. 1976. Seasonal polyphenism. Evolutionary Biol. 9:259-333. Owen, D.F. 1971. Tropical Butterflies: the ecology and behavior of butterflies in the tropics with special Tauber, M.J., Tauber, C.A. & Masaki, S. 1986. Seasonal reference to African species. Clarendon Press. Adaptation of Insects. Oxford: Oxford University Oxford. Press. Pinratana, B.A. 1988. Butterflies in Thailand 6: Windig, W.W.1992. Seasonal polyphenism in Bicyclus Satyridae, Libytheidae and Riodinidae. Viratham safitza, a continuous reaction norm. Netherl. J. Press, Bangkok. p. 60. 44 colour plates. Zool. 42:583-594. Rienks, J.H. 1985. Phenotypic response to photoperiod and temperature in a tropical pierid butterfly. Aust. J. Zool. 33:837-847.
Manuscript received on 10.06.2010, Accepted on 07.03.2011 Univ.Parasitic j. zool. infestation Rajshahi. inUniv. laboratory Vol. 29, rat2010 strain pp. 41-46 ISSN 1023-61041 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Parasitic infestation in laboratory rat strain, Long-Evans (Rattus norvegicus Berkenhout, 1769) Md. Abdul Gofur1, Hamida Khanum1,Milka Patracia Podder1 and Zaibun Nessa2 1Department of Zoology, University of Dhaka, 2Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka
Abstract: In the present investigation, 30 Long-Evans were examined. Out of 30, 23 rats were infected by helminth parasites, two species of helminth parasites from two taxonomic groups were identified, cestode Hymenolepis nana and nematode Syphacia muris. The prevalence and intensity of parasites were quite different in different groups of helminth parasites in hosts. The prevalence and intensity of infestation of cestodes were 26.67% and 5.57±1.5 while higher of nematodes 66.67% and 60.85±8.1 in the Long-Evans respectively. Hymenolepis nana was found in small intestine and Syphacia muris in all parts of the alimentary tract. The prevalence and intensity of infestation was 36.67% and 5.09±1.6 respectively in small intestine. The prevalence of infestation was similar both in large intestine and caecum (63.33%), and in rectum (63.33%). The intensity of infestation was 24±3.30 and 39.16±4.10 in large intestine and caecum, and in rectum respectively.
Key words: Long-Evans, Rattus norvegicus, cestodes, nematodes, prevalence, intensity.
Introduction they contaminate human food and clothing both with their feces and urine and also by physical Helminthes parasitized rats are studied to identify contact. This behaviour is enough to lend support the species and for determination of prevalence to the idea that they not only maintain but also and intensity of helminthes (Stanley & Virgin, disperse some of the human parasites as well as 1993). Mice and rats are the most common diseases. laboratory animals used in research and testing. Long-Evans is one of the important strains of Various diseases caused by helminth parasites laboratory rat Rattus norvegicus, Berkenhout, are significant health problem in both veterinary 1769, harbored in most of the animal houses in and human medicine. Nematode parasites alone Bangladesh. Throughout the world, necessary are estimated to produce infections over more steps have been taken in order to detect and than three millions farm and pet animals in the identify the parasites of laboratory animals to keep United States (Wiroreno, 1978). Both domestic them physically fit by controlling or eradication of and wild animals are subject to a variety of parasites. parasites. Cestode and nematode parasites in rat have been reported from all parts of the world. Parasitic studies on rats and mice have been Vampirolepis nana and Hymenolepis diminuta are done in most countries and reported that rats and commonly found in rats and mice and potentially mice harbor a number of helminth parasites such transmissible to man (Jawdat & Mahmoud, 1980). as trematodes, cestodes, nematodes and acanthocephalan (Singh 1962, Gupta & Trivedi, The occurrence of Hymenolepis diminuta, 1985). In developing countries, where conditions Vampirolepis nana in certain rodents is of interest are more suitable for these hosts where since the possibility exists that rats and mice may awareness to control them is less than to that of served as reservoir hosts and aid in the the developed countries this information is of dissemination of these worms to domestic animals particular importance because of the role which and man (Jawdat & Mahmoud, 1980), causing they have as reservoir hosts of some parasites zoonoses. Spindler (1930) recovered Vampirolepis infecting man. nana in 3.6% of 2152 people examined from South-West Virginia. Senekiji et al. (1940) recorded The presence of rats and mice therefore, that the incidence of Vampirolepis nana was 2.1% represents a potential epizootiological problem of 1000 people examined in Iraq. Salem et al. (Spatafora & Platt, 1982). The rats and mice live (1968) reported that the presence of Vampirolepis very intimately with humans and ubiquitously nana was 42.4% of 204 young people from Mosul distributed in natural environment (Seo et al. area of Iraq. 1981). In addition, during the daily promenade, 42 Gofur et al.
Infection with Syphacia muris shows symptoms (Siebold, 1852) and nematode Syphacia muris which are poor condition, rough hair coats, reducer (Yamaguti, 1935). growth rate, rectal prolapsed (Hoag, 1961). Experimentally Syphacia muris infected animals grew slower than uninfected animals (Wagner, 1988). Increase in resistance of pinworm infection with advancing age of rats (Wagner, 1988). Pinworms of laboratory rodents are generally not considered pathogens (Flynn, 1973). In Bangladesh, very few studies have been carried out on identification of the parasites and worm burden of rats and mice. These include the work of Huq (1969), Shaha (1974), Bhuiyan et al. (1996), Khanum et al. (2001), Khanum et al. (2009), Alam et al. (2003), Khanum & Arefin (2003) and Muznebin et al. (2009). The present study was undertaken to identify prevalence and Fig. 1. Scolex of Hymenolepis nana(40×10) intensity of helminth parasites of the Long-Evans (Rattus norvegicus Berkenhout, 1769). Materials and Methods The hosts were reared in cages and special type of food and water were supplied. Food materials supplied for the rearing of long-evans were rice polish, wheat bran, maize, protein, soyabean pulses, coarse flour, salt, soyabean oil, g.s. vitamin, treacle etc. After measuring the length and weight of the hosts, the parasites were collected. The cestodes were dipped quickly in a dish of hot fixation (AFA) when the scolices were in extended Fig. 2. Anterior portion of Syphacia muris(40×10) condition. After 10 minutes cestodes were then preserved in 70% ethyle alcohol. Cestodes were cleared and accomplished more rapidly when Lacto-phenol was used. Worms with thick cuticle required over night to be fairly cleaned. The nematodes were washed thoroughly in the saline solution and were cleared in Lacto-phenol. Finally the nematodes were preserved in 70% ethyle alcohol. The parasites were identified following the key given by Yamaguti (1959 and 1961). Like other helminthes nematodes were cleared and studied satisfactorily as temporary whole mount in Lacto-phenol. The smaller forms required overnight and the larger forms required more than 7 days to be fairly cleaned. Fig. 3. Posterior portion of female Syphacia muris(40×10) Results Prevalence and intensity of helminth parasitic A total of 1256 helminth parasites were collected infestation in male and female hosts: from 30 hosts, Long-Evans. Each worm was examined for taxonomic identification. Two The prevalence of helminth parasites infection was species of helminth parasites were identified from 76.67% and the percentage intensity of infestation two taxonomic groups, Hymenolepis nana was 54.60±7.3 in Long-Evans. The prevalence of Parasitic infestation in laboratory rat strain 43
infestation in male was 80% and in female was comparatively higher (63.17±9.2) in male and lower 73.33% whereas the intensity of infestation was (45.27±6.60) in female, (Table-1).
Table 1. Prevalence and intensity of infestation of helminth parasites according to sex of the hosts. Sex of the No. of hosts No. of hosts Prevalence Total no. of Intensity Host host examined infected (%) parasites collected (±SD) Male 15 12 80 758 63.17±9.2 Rattus Female 15 11 73.33 498 45.27±6.60 norvegicus Total 30 23 76.67 1256 54.60±7.3
The percentage of infection varied from host to were higher than the cestodes. Intensity of host and also parasite to parasite. The prevalence cestodes and nematodes were 5.57±1.5 and of cestodes and nematodes were 26.67% and 60.85±8.1 respectively, (Table-2). 66.67% respectively. Prevalence of nematodes
Table 2. Prevalence and intensity of infestation of different groups of parasites. Total no. of Total no. of Prevalence Total no. of Intensity Groups hosts examined hosts infected (%) parasites collected (±SD) Cestodes 30 8 26.67 39 5.57±1.5 Nematodes 30 20 66.67 1217 60.85±8.1
The prevalence of each species of helminth The intensity of H. nana was higher (8.5±2.1) in parasite in male and female slightly varied but male than in female (3.67±1.1). But the intensity percentage intensity greatly varied. The of S. muris was higher (73.54±10.7) in male than prevalence of H. nana was higher (40%) in female in female (45.33±5.40), (Table-3). than in male (13.33%). But the prevalence of S. muris was higher (73.33%) in male than in female (60%).
Table 3. Prevalence and intensity of each species of helminth according to sex of the hosts. Sex of Name of the Total no. of Total no. of Prevalence Total no. of Intensity the hosts parasites hosts examined hosts infected (%) parasites collected (±SD) Hymenolepis nana 15 2 13.33 17 8.5±2.1 Male Syphacia muris 15 11 73.33 809 73.54±10.7 Hymenolepis nana 15 6 40 22 3.67±1.1 Female Syphacia muris 15 9 60 408 45.33±5.40
The occurrence of parasites was most abundant caecum and the rectum (63.33%). The lower in large intestine and caecum and in rectum. No prevalence was recorded in small intestine parasites were found in esophagus and stomach. (36.67%). So the prevalence in esophagus and stomach is Comparatively lower intensity of infestation was absent. 11 (36.67%) parasites were collected from small intestine, 19 (63.33%) parasites were found in small intestine (5.09±1.6). The higher collected from both the large intestine, caecum intensity was found in rectum (39.16±4.10), and the rectum. The highest prevalence of slightly higher intensity was found in large infestation was found in both the large intestine, intestine and caecum (24±3.30), (Table-4). 44 Gofur et al.
Table 4: Prevalence and intensity of organ wise helminth parasites in Long-Evans. Total no. of Total no. of Total no. of Intensity Location Prevalence (%) hosts examined hosts infected parasites collected (±SD) Esophagus 30 0 0 0 0 Stomach 30 0 0 0 0 Small Intestine 30 11 36.67 56 5.09±1.6 Large Intestine Caecum 30 19 63.33 456 24±3.30 Rectum 30 19 63.33 744 39.16±4.10
The prevalence and intensity of cestodes in small whereas in large intestine and caecum were 60% intestine 26.67%, 3.75±1.1, and in large intestine and 25±3.4 respectively. The cestode was also and caecum were 3.33%, 6±1.8. The cestode in absent in rectum, whereas the prevalence and esophagus and stomach was absent. In small intensity of nematodes were highest in rectum intestine, the prevalence and intensity of 63.33% and 39.16±4.10 respectively, (Table-5) nematodes were 10% and 8.67±1.9 respectively,
Table 5: Prevalence and intensity of different groups of parasites in Long-Evans. Cestodes Nematodes No. of the No. of No. of No. of No. of Organs hosts Prevalence Intensity Prevalence Intensity hosts parasites hosts parasites examined (%) (±SD) (%) (±SD) infested collected infected colleted Small Intestine 30 8 26.67 30 3.75±1.1 3 10 26 8.67±1.9 Large Intestine 30 1 3.33 6 6±1.8 18 60 450 25±3.4 & Caecum Rectum 30 0 0 0 0 19 63.33 744 39.16±4.10
Discussion The prevalence of helminth parasites in male was 80% and in female was 73.33%. But the intensity In the present study, the data showed that the in male was higher (63.17(9.2) than in female overall prevalence of infestation in Long-Evans (45.27(6.60). Bhuiyan et al. (1996), reported that was 76.67%. Density dependent effects on the among the three species of rodents such as establishment and growth of H. diminuta have Bandicota bengalensis, Rattus rattus and M. been reported by (Chandler, 1939). In general musculus, the prevalence of infection was higher there was an inverse relationship between worm in female than male. Khanum et al. (2001), size and worm burden, termed the `Crowding observed the incidence of helminth infection and effects` (Read, 1951). Similar relationship was the comparative efficacy of a single 400mg dose observed between worm fecundity and worm of Albendazole, 600 mg dose of Mebendazole burden (Hager, 1941). Density dependent effects and Neem extract (Azadirachta indica) in the both worm dry weight and egg production and at treatment of roundworm, hookworm and highest dose both dry weight and egg production whipworm infestations. Khanum & Arefin (2003), decreases significantly (Quinnell, 1988). Chappel reported that the prevalence of infestation of & Pike (1976) have reported a small difference in helminth groups were almost similar with slightly the rate of worm rejection and in worm size higher prevalence of cestodes in Swiss albino between adult and juvenile rats which were mice. Khanum et al. (2009), reported that the slightly greater and slightly smaller respectively in prevalence of infection was found highest in the adults. caecum (95.83%) and intensity of infection was Huq et al. (1985), reported 96.41% prevalence in highest in anterior part of intestine. From the Rattus rattus . Bhuiyan et al. (1996), reported that present result it appears that the cestodes and about 83.33% Bandicota bengalensis and 82.08% nematodes are most common in laboratory rats Rattus rattus were infected with helminth and mice, due to its special type of feeding parasites in Bangladesh. In the present study, the habits. intensity of infestation in Long-Evans was found The feeding habit, non-sterilized water and food 54.60±7.3. supply, overcrowding, inadequate ventilation and improper handling by lab attendant may be some Parasitic infestation in laboratory rat strain 45
contributing factors for high worm burden in Long- Khanum H, Chowdhury S, & Sen A. 2001. Comparative Evans in the present animal house. Such an efficacy of Albendazole, Mebendazole and neem leaf extract in the treatment against human intestinal infected individual causes zoonotic disease and helminth. Trans. Zool. Soc. East. India. 5(1):65-69. also remains completely unfit for use in any kind of scientific work. But throughout the world Long- Muznebin F, Khanum H, Nessa Z, & Islam D. 2009. Endoparasitic Infection and Histopathological Evans are widely used for experimental works. Effects in Laboratory Rat Strain, Long-Evans The result in the present study emphasized that (Rattus norvegicus Berkenhout, 1769). Bangladesh the living condition of the animal house belonging J. Sci. Ind. Res. 44(1):109-116. to the IFST of BCSIR should be improved. Quinnell, R.J. 1988. Host age and the growth and References fecundity of Hymenolepis diminuta in the rat. J. Helminthol. 62:158-162. Alam, M.S., Khanum, H. & Zaibunnesa 2003. Helminth infection in laboratory rat strain, Long-Evans (Rattus Read, C.P. 1951. The “Crowding effects” in tapeworm norvegicus Berkenhout, 1769). Bangladesh J. Zool. infections. Parasitol. 37:174-178. 31(2):221-225. Salem, H., Hayatee, G., Awanees, A.M. & Al-Allaf, G. Bhuiyan, A.I., Ahmed, A.T.A. & Khanum, H. 1996. 1968. Evolution of the anthelmic activity of Endoparasitic Helminths in Rattus rattus Linnaeus & Thiobenolozole in man. J. Trop. Med. Hyg. 71:1-7. Bendicota bengalensis Gray. J. Asiat. Soc. Bangladesh, Sci. 22:189-194. Senekiji, H.A., Boswell, C. & Beattie, C. 1940. The incidence of intestinal parasites in Iraq. Trans. Roy. Chandler, A.C. 1939. The effects of number age of worms Soc. Trop. Med. Hyq. 33:349-352. on development of primary and secondary infections with Hymenolepis diminuta in rats and an investigation Seo, B.S., Cho, S.Y., Hong, S.T., Hong, S.J. & Lee, into the true nature of ‘Premonitions’ in tapeworm S.H. 1981. Studies on parasitic helminthes of Korea. infection. American J. Hygiene. 29:105-114. J. Parasitol. 19(2):131-136. Chappell, L.R. & Pike, A.W. 1976. Loss of H. diminuta Shaha, J.G. 1974. Taxonomy of some of the helminthes from the rat. International J. Parasitol. 6:333-339. of house rats, house mice and house shrews of Flynn, R.J. 1973. Nematode Parasites of Laboratory Dacca city. M. Sc. Thesis. University of Dhaka, Animals, Iowa State University. Press, Ames Iowa. Bangladesh. 120 pp. pp. 203-320. Singh, K.S. 1962. Parasitological survey of Kumaun Gupta, S.P. & Trivedi, K.K. 1985. Nematode parasites region. Part XI. Four nematodes from the rat, Rattus of vertebrates. A new nematode, Gongylonema fotedari spnov (Family: Spiruridae Oreley, 1985) norvegicus. Indian J. Helminthol. 14(2):98-111. from Indian mole rat Bandicota bengalensis from Spatafora, G.A & Platt, T.R. 1982. Survey of the Lucknow, U.P. Indian J. Helminthol. 37(11):100-108. Helminth Parasites of the rat, Rattus norvegicus, Hager, A. 1941. Effects of dietary modifications of host from Maymont Park, Richmond, Virginia. Virg. J. rats on the tapeworm Hymenolepis diminuta. Iowa Sci. 32:3-6. State College. J. Sci. 15:127-135. Spindler, L.A. 1930. On the occurrence of the tapeworm Hoag, W.G. 1961. Oxyuriasis in laboratory mouse (Hymenolepis diminuta) and the dwarf tapeworm colonies. Am. J. Vet. Res. 22:150- 153. (Hymenolepis nana) in South-West Virginia. J. Huq, M.M. 1969. A survey of the helminth parasites of Parasitol. 16:38-40. roof rats Rattus rattus, digger rats Bendicota bengalensis and mole Scalopus scalopus in Stanley, S.L.Jr. & Virgin, I.V.H.W. 1993. Scid mice as Mymensingh district, East Pakistan, Pak. J. Vet. Sci. models for parasitic infections. Parasitol. Today. 3:65-68. 9:406-411 Huq, M.M., Karim, M.J. & Sheikh, H. 1985. Helminth Wagner, M. 1988. The effect of infection with the parasites of rats, house mice and moles in pinworm (Syphacia muris) on rat growth. Lab. Anim. Bangladesh. Pak. J. Vet. Sci. 5(3):143-144. Sci. 38:476-478. Jawdat, S.Z. & Mahamoud, S.N. 1980. The incidence of Wiroreno, W. 1978. Nematode parasites of rats in cestoidean and acanthocephalan parasites of some West-Java, Indonesia. South-East Asian J. Trop rodents in Iraq. Bull. Nat. Hist. Res. Centre. 7(4):55- Med Public Health. 4:520. 71. Khanum, H. & Arefin, N. 2003. Helminth Burden in Yamaguti, S. 1959. “Systema Helminth”. Vol. II. Laboratory mice, Mus musculus. Bangladesh J. Cestodes of Vertebrates. Inter. Science Publisher Zool. 31:117-123. Inc., New York. 860 pp.. Khanum H, Muznebin, F. & Nessa Z. 2009.Nematode Yamaguti, S. 1961. “Systema Helminth”. Vol. II. and Cestode prevalence, Organal distribution and Nematodes of Vertebrates. Inter Science Publisher histological effects due to parasitic infection in Inc., New York. 1261 pp. Laboratory rat strain, Long-Evans (Rattus norvegicus Berkenhout, 1769). Bangladesh J. Sci. Ind. Res. 44(2):207-210.
Manuscript received on 13.07.2010, Accepted on 05.01.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 47-50 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Seasonal occurrence of parasites of the major carp, Cirrhina mrigala (Hamilton) collected from Rajshahi, Bangladesh Abdus Salam Bhuiyan*, Sarmin Akther and Noor-E-Amena Fisheries Research Laboratory, Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh *Corresponding author
Abstract:The research work was conducted to find out the seasonal occurrence of parasites of the major carp, Cirrhina mrigala from different water bodies of Rajshahi district during April 2009 to January 2010. A total of 95 fishes were examined of which 72 fishes were infected. Thirteen species of parasites viz. Trichodina pediculatus, Chilodonella cyprini, Ichthyophthirius multifiliis, Apiosoma sp., Dactylogyrus vastator, Gyrodactylus elegans, Hirudo sp., Argulus foliaceus, Lernaea sp. Phyllodistomum agnotum, Eucreadium sp., Camallanus ophiocephali and Pallisentis sp. were recorded from the infected fishes. Among all the parasites G. elegans was the most dominant parasite and skin had been found as the most vulnerable site for parasitic infestation. The maximum infestation was observed in winter season and the minimum infestation was observed in rainy season.
Key words: Cirrhina mrigala,parasite, prevalence, abundance, mean intensity, infestation.
Introduction Materials and Methods Bangladesh is gifted with fisheries resources. The Period and methods of sample collection soil, water and climate are very much suitable for The investigation was carried out from April 2009 to aquaculture and fisheries resources management. January 2010. To observe the seasonal infestation, So, major carp like C. mrigala is one of the most the study period was divided into three remarkable commonly cultured indigenous freshwater fish in seasons. They were summer (April-June), rainy Bangladesh. It is highly nutritious, delicious and (July-October) and winter (November-January). has high market value and its fry and fingerlings Live or freshly dead fishes were collected every are easily available. week at a regular interval from different fish Nowadays diseases have become a major markets of Rajshahi city. The collected fishes were problem in fish production. The fish parasites may kept in the shopping bags and transported to the cause fish mortality in culture fisheries where the Fisheries Research Laboratory, Department of entire population of pond may be killed, resulting Zoology, University of Rajshahi. in loss of potential food and economic loss to the Laboratory technique culturists (Sood, 1698). After collection, all the host fishes were examined The success of the implementation of various as early as possible, generally within 12 hours. fishery development programs depends to a great External parasites from body surface, fin and gill extent on the intensification of the fish were removed by scrapping the slime on a clean parasitological research, as the improvement of dry glass slide and spread it evenly. After that a fish yield can mainly be achieved from healthy fish cover slip was placed over the materials. The gill stock (Srivastava, 1975). So, proper management arches were removed and macerated under a would contribute and play supporting role to compound binocular microscope. In case of harness the maximum benefit from fisheries monogeneans the gills were removed to Petri resources. dishes containing water and gently scrapped to dislodge monogeneans. For endoparasites, fishes C. mrigala is a very expensive fish. It is very were dissected out ventrally by a sharp scalpel to widely cultured in Bangladesh. The farmers very observe parasites inside buccal cavity, stomach often face the problems of parasites in it. The and intestine. The whole gut was removed in a present work has been undertaken with a view to watch glass containing normal saline solution and knowing the seasonal occurrence of parasites in search under microscope for the protozoan and C. mrigala of Rajshahi. helminth parasites. 48 Bhuiyan et al.
Identification of parasites Results and Discussion Ectoparasites and endoparasites were identified A total of 95 samples were examined from which on the basis of available taxonomical characters a total of 72 (75.79%) fishes were found infected. and figures as described by Yamaguti (1963), Thirteen species of parasites were identified of Kennedy (1975), Kabata (1985), Willhelm which nine were ectoparasites and four were Schaperclaus (1991), Hafizuddin and endoparasites. The result is presented in tables- Shahabuddin (1996) and Dr. Kirtunia Juran 1,2,3 and Fig.1. Chandra (2004) etc.
Table-1. List of parasites and their site of infestation. Host fish Parasites Site of infestation Protozoa : Trichodina pediculatus Gill Chilodonella cyprini Skin Ichthyophthirius multifiliis Skin, gill and fin Apiosoma sp. Skin and fin Monogenea : Dactylogyrus vastator Gill Gyrodactylus elegans Skin C. mrigala Digenea : Phyllodistomum agnotum Stomach Eucreadium sp. Intestine Acanthocephala : Pallisentis sp. Intestine Nematoda : Camallanus ophiocephali Intestine Annelida : Hirudo sp. Skin, fin and gill Crustacea : Argulus foliaceus Skin and fin Lernaea sp. Gill
Protozoans and monogeneans were very from stomach and intestine. The observation of common on the gill, skin and fin of host fishes. Chandra (1985) and Farhaduzzaman (2005) were Crustaceans were mostly found abundantly also similar with this result. The present result is attached on the fin and skin. On the other hand also similar with that of Bhuiyan et al. (2006). digenean flukes and nematodes were recorded
Table-2. Organ wise distribution of parasites in C. mrigala. Number of parasites in different organs Recovered Parasites Skin Fin Gill Intestine Stomach Total D. vastator 0 0 76 0 0 76 G. elegans 93 0 0 0 0 93 C. cyprini 24 0 0 0 0 24 Apiosoma sp. 17 29 0 0 0 46 I. multifiliis 24 15 28 0 0 67 Hirudo sp. 08 05 02 0 0 15 T. pediculatus 0 0 35 0 0 35 A. foliaceus 77 52 0 0 0 129 Lernaea sp. 0 0 25 0 0 25 C. ophiocephali 0 0 0 52 0 52 Eucreadium sp. 0 0 0 29 0 29 Pallisentis sp. 0 0 0 37 0 37 P. agnotum 0 0 0 0 80 80 Total 243 101 166 118 80 708
The parasites were mainly found in different skin. The lowest prevalence, abundance and organs like skin, fin, gill, intestine and stomachs of mean intensity were found at stomach and only the host. A number of 708 parasites of 13 species one species (Phyllodistomum agnotum) was were collected from these organs. recorded there. The highest prevalence, abundance and mean intensity of the parasites were recorded from the Parasites of the major carp, Cirrhina mrigala 49
Prevalence (%) Abundance Mean intensity 120
100
80
60 Values 40
20
0 April May June July August September October November December January
2009 2010 Months
Fig. 1. Monthly prevalence, abundance and mean intensity of parasites of C. mrigala.
During the study period the highest (138) Bhuiyan, A.S., Musa, A. S. M., Musa, G. M. & Zaman, T. parasites were recorded in January and the 2006. Occurance of parasites in three indigenous second highest value (95) was found in major carps. Bangladesh J. Zool. 36(2):229-235. December. The lowest value (41) was recorded in Bhuiyan, A.S. & Musa. A.S.M. 2008. Seasonal September .The highest (100%) prevalence of prevalence and intensity of infestation by the parasites was recorded in January and the lowest ectoparasites in carp relating to physio-chemical value (55.55%) was recorded in April. The highest parameters in some ponds of Mymensingh and abundance value (13.80) was recorded in January Bogra districts of Bangladesh. Bangladesh. J. Sci. and the lowest (4.55) was recorded in September. Ind. Res. 43(3):411-418. The highest mean intensity of parasites was Chandra, K.J. 1985. Incidence and intensity of infestation observed as 13.80 in January and the lowest as of Pallisentis ophiocephali (Thapar) on the host 6.83 in September. Channa punctatus (Bloch). J. Asiatic Soc. Bangladesh (Sci.) 11:47-54. The highest prevalence (92.59%), abundance (10.03) and mean intensity (11.64) were observed Chandra, K.J. 2004. Fish Parasitology. Lima printing in winter. The lowest prevalence (71.05%), press. Mymensingh. p. 176. abundance (5.74) and mean intensity (8.07) were Chandra K.J. Islam K.Z. & Wootten. R. 1997. Some recorded in rainy season. aspects of association and development of Lytocestus indicus moghe in catfish, Clarias So, in general it can be said that the infestation batrachus. Bangladesh J. Fish. Res., 1:31-38. was maximum in winter season and minimum in rainy season. Similar findings were also noticed Farhaduzzaman, A.M. 2005. Parasites of an Indian Major Carp, Labeo rohita (Hamilton 1822). by Banu et al. (1993), Hossain et al. (1994), Bangladesh, J. Aquacult. 2:50-60. Akhter et al. (1997), Chandra et al. (1997), Bhuiyan et al. (2008) and Hossain et al. (2010). Hafizuddin, A.K.M. & Shahabuddin, M. 1996. Parasitic monogeneans from some freshwater fishes of References Comilla, Bangladesh. Chittagong Univ. Stud. Sci. 20:113-126. Akhter, M., D'silva, J. & Khatun, A. 1997. Helminth parasites of Anabas testudineus (Bloch) in Hossain, M.A., Banu, A.N.H. & Khan, M.H. 1994. Bangladesh. Bangladesh J. Zool. 25:135-138. Prevalence of ectoparasites in carp nursery operation of greater Mymensingh. Progress. Banu, A. N. H., Hossain, M. A. & Khan, M. H. 1993. Agricult. 5:39-44. Investigation into the occurrence of parasites in carps, catfish and tilapia. Progr. Agricult. 4:11-16. 50 Bhuiyan et al.
Hossain, M.D. & Hossain M.K. 2010. Prevalence of ectoparasites of carp fingerlings at Santaher, Schaperclaus, W. 1991. Fish Diseases. Vol. 1 and 2. Bogra. Abstract of seminar on fisheries activities in Oxonian Press Pvt. Ltd. New Delhi. northern Bangladesh: sustainable livelihood and Sood, M.L. 1968. Some nematode parasites from climate change aspects. Department of Fisheries freshwater fishes of India. Ind. J. Helm. 20(2):83- University of Rajshahi. 27p. 110. Kabata, 1985. Parasites and Diseases of Fish cultured Srivastava, C.B. 1975. Fish pathological studies in in Tropics. Taylor and Francis Ltd. 318p. India, a brief review. B.S. Chauhan Comm. 349- Kennedy, C.R. 1975. The natural history of Slapton Bay 358pp. Natural Reservoir. VIII: The parasite of fish with Yamaguti, S. 1963. Systema helminthum. Vol. IV. special reference to their use as source of Monogenea and Aspidocotylea. Interscience information about the aquatic community. Fiel. publishers. N.Y. 699p. Stud. 4:177-189.
Manuscript received on 05.12.2010, Accepted on 01.03.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 51-56 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Toxicity of dishwashing liquids against the American cockroach, Periplaneta americana L. (Dictyoptera: Blattidae) A.M. Saleh Reza, Md. M.I. Din and Selina Parween* Department of Zoology, University of Rajshahi,Rajshahi-6205, Bangladesh *Corresponding author. e-mail: [email protected]
Abstract: Dishwashing liquids viz. Vim and Trix at concentrations of 0.5, 1.0, 1.5 and 2.0% were toxic against the adult American cockroach, Periplaneta americana L. after 24-h of exposure. Knockdown effect of the detergents was found to be 70-78% by contact with both the detergents. Whereas, by dipping 100% of the cockroaches were knocked down and ultimately died at 24-h exposure. The LC50 values were calculated for contact toxicity were 1.490% for Vim and 0.883% for Trix. The results revealed that Trix is more toxic than Vim against the adult P. americana. Keywords: Dishwashing liquid, toxicity, Periplaneta americana.
Introduction or detergent toxicity on agricultural pest insects are available, but such reports against the Cockroaches are the most common household cockroaches are rare. pests. Among the cockroaches, the American cockroach, Periplaneta americana L. is quite big This work was aimed at determining the toxicity of in size with large wings, and are common in the the most commonly used liquid dishwashing tropics. detergents, Vim and Trix against the adults of P. americana. Cockroach control program has used every class of insecticides. Each time a new group of Materials and Methods insecticide is discovered, its potentiality is tested The experiment was conducted at the laboratory of against cockroaches (Szumlas, 2002). With the the Department of Zoology, University of Rajshahi, advent of time, cockroaches developed resistance against most of the chemical insecticides. Besides Bangladesh, at room temperature (25-28°C). Adult that the negative impacts of the insecticides on cockroaches were collected from different sources the environment and human health, bound the with the help of scoop net, then acclimatized them scientists to search for safer, potential and more to the laboratory conditions keeping in glass economical alternatives for cockroach control. beakers covered by fine meshed cloth, and providing pieces of fresh bread as food. After 48 The use of soap to control insects is not a new hours, healthy adults were separated to use in the concept. It is reported that in 1787 soap was first experiment. The experimental roaches were not used to control small and soft-bodied insect pests sexed and their age varied form 5-7 days. of plants, e.g., aphids, thrips, psyllids, whiteflies, scales and even mites (Moore et al., 1979). Ware Detergents used: The commonly available liquid (2000) reported that soaps are potentially toxic dishwashing detergents, Vim and Trix were used against the slow moving arthropods. Soaps and in this experiment. Vim is a product of Unilever detergents act strictly as contact insecticides Bangladesh Ltd., and Trix is manufactured by which have no residual effect (Szumlas 2002). Reckett Benckiser (Bangladesh) Ltd. Vim is Abbasi et al. (1984) commented that composition whitish and Trix is lemon green in colour. of the soaps which were used in the past, was The concentrations of detergents (0.5, 1.0, 1.5 different from that of the presently available and 2.0%) both were prepared in tap water just soaps, and the present day soaps might be before setting of the experiment. Two methods of significantly more effective against a wide range detergent application were adopted: (i) contact of insect groups. They reported that the soft soaps method and (ii) dipping /immersion method. The (fatly acid with potassium salts) produced 100% adult cockroaches used for the experiment were mortality in the German cockroach at only 2% kept in beakers (500ml) for three hours before the concentration. Published literature on liquid soap start of experiment. 52 Reza et al.
A. Knock down effect of Vim and Trix experiment was run simultaneously with six cockroaches keeping them in similar but untreated i) Contact treatment: Plastic containers of 20cm plastic containers. The experiment was replicated height and 15cm diameter were used as treatment for five times. arenas. Inside wall of the containers at a height of 15cm were lubricated with petroleum jelly to B. Toxic effect of Vim and Trix prevent escape of the cockroaches. Bottom and The acute toxicity of the detergents was the inside walls of the container (up to 15cm high) determined from the contact treatment only. The were thoroughly brushed with detergent solution, treatment was done similarly as mentioned for the six cockroaches were released inside, and the knockdown experiment. The same concentrations mouth was covered with a fine meshed cloth. as mentioned were used for each of the ii) Dipping/immersion treatment: In 500ml glass detergents. Twelve adult cockroaches were used beakers the liquid detergent solutions were for each concentration of each detergent. A poured up to 300ml. Six cockroaches were dipped control experiment was kept with 12 cockroaches into the solution until they all were completely wet. as well. These cockroaches were then transferred to Number of dead cockroaches was counted after plastic containers (20cm height × 15cm diameter) 1, 3, 6 and 12 hour of exposure. The dose covered with fine mesh cloth, and the upper rim mortality data were analyzed using Probit Analysis (5cm) of the inner wall of the container was (Busvine, 1971) to determine the LC value of coated with petroleum jelly to avoid escape of the 50 both Vim and Trix against P. americana. cockroaches. Results and Discussion Data Collection: The treated cockroaches were kept under continuous observation for nearly 15 A. Knockdown effect of Vim and Trix hours to determine the immediate or delayed The knockdown data of Vim and Trix are given in knockdown effects of the detergents. Lack of Table 1 and 2 for contact and dipping/immersion movement of legs and antennae, and absence of methods respectively. The contact action of Trix natural response were considered as the against the adult cockroach was greater than that characters for knockdown effects. No attempt was of Vim. Total number of dead cockroaches was made to awaken the cockroach by prodding. The 125 in Trix which was only 82 in vim treatment. knockdown cockroaches were counted 1, 3, 6, 12 The knockdown effect was increased with the and 24 hours after treatment (HAT), and were increase of concentration of the detergents and converted into percentages. with the increase of treatment durations. Vim at For knockdown durations six adult cockroaches 0.5% concentration showed no effect up to 3h, were used for each concentration of each whereas, knockdown effect was observed from detergent for each treatment. A set of control the first hour of Trix treatment (Table 1).
Table 1. Knockdown duration of adult P. americana by contact toxicity of liquid dishwashing detergents (Number of cockroach at each concentration of each treatment- 60).
Knockdown duration (HAT) Detergent Concentration (%) Total nos. died % dead 1 3 6 12 24 Control 0.0 00 00 0 00 00 0 00.00 0.5 00 00 01 01 02 04 06.67 Vim 1.0 01 02 03 03 06 15 25.00 1.5 02 03 04 05 07 21 35.00 2.0 06 08 08 09 11 42 70.00 0.5 01 01 02 03 05 12 20.00 Trix 1.0 03 04 05 06 08 26 43.33 1.5 05 07 08 09 11 40 66.67 2.0 06 08 10 11 12 47 78.33
Toxicity of dishwashing liquids against the cockroach 53
The immersion method was found to be equally after 1hr, which was increased to 12 after 24h. effective for all concentrations of both Vim and Trix Whereas, the higher concentrations of both the (Table 2). At 0.5% concentration of Vim, the detergents knocked down 10-12 cockroaches at number of knockdown cockroach was 6 after 1h, first hour, and 100% mortal effect was observed which increased to 12 after 24h. The same afterwards (Table 2). concentration of Trix knocked down 5 cockroaches
Table 2. Knockdown duration of adult P. americana after dipping in liquid dishwashing detergents (Number of cockroach at each concentration of each treatment- 60).
Knockdown duration (HAT) Detergent Concentration (%) Total nos. died % dead 1 3 6 12 24 Control 0.0 00 00 0 00 00 0 00.00 0.5 06 06 07 10 12 41 68.33 Vim 1.0 12 12 02 21 13 60 100 1.5 10 12 30 02 06 60 100 2.0 20 12 14 22 - 60 100 0.5 05 07 09 10 12 43 71.67 Trix 1.0 12 12 10 12 14 60 100 1.5 14 12 21 12 01 60 100 2.0 20 04 12 11 13 60 100
These results revealed that dipping of both Vim and Trix at concentrations of 1.5-2% cockroaches in the liquid detergent affected them immobilized 16-33% adult American cockroaches more than that caused by the contact to the one hour after dipping treatment, while by contact, detergents. Concentration of 2% caused 100% to only 10% of the roaches were affected at a suffer and ultimate death. Higher concentrations concentration of 2% of the detergents at the same of these detergents would be effective in exposure period. immobilizing cockroaches more rapidly. Though knockdown effect was more in dipping B. Toxic effect of Vim and Trix treatment than contact treatment of both Vim and Trix, but the effect was more in contact treatment The mortality data revealed that the contact action than dipping treatment in both the detergents. of Trix is greater than that of Vim. Toxicity was Whereas, dipping into the detergents were highly observed to be increased with the increase of lethal for the roaches. After 12hr contact, the LC concentrations of the detergents as well as 50 values were obtained as 1.49% (Vim) and 0.88% increase of exposure. The LC values of Vim 50 (Trix), but no mortality was resulted when the were obtained as 2.10, 1.75, 1.7 and 1.49% after cockroaches were dipped into the detergents until 1, 3, 6 and 12 hours exposure respectively (Figure they were fully soaked. 1). These values for Trix were determined as 1.93, 1.39, 1.08 and 0.88% after 1, 3, 6 and 12 Abbasi et al. (1984) and Ware (2000) suggested hours exposure respectively (Figure 1). The that soap solutions may block spiracles of insects results revealed that both Vim and Trix are highly thus interfering with their respiration that results in toxic against the adult American cockroach. asphyxiation and ultimate death. Cockroaches possess an oily film and a waxy cuticular Discussions hydrocarbon layer throughout on their body surface The commercial liquid dishwashing detergents which extends into their tracheae to prevent water have been reported to be toxic against the loss (Cornwell, 1968). Szumlas (2002) advocated cockroaches. Szumlas (2002) used 0.05-5.0% that dipping in liquid soaps may interact with that concentrations of Dawn Ultra liquid dishwashing hydrophobic cuticle layer and quickly spread over detergent (Proctor & Gamble Co.) to control adults the body surface of the cockroach, entering into the and nymphs of the German cockroach Blatella spiracles and tracheae. So, the liquid soap may act germanica L. and reported that dipping treatment as a physical plug into the tracheae and block the (≥3%) caused an immediate knockdown followed exchange of air resulting in quick mortality at low by 100% quick death. In the present experiment, concentrations. 54 Reza et al.
1 hr exposure 1 hr exposure 6 6 5 5 4 4 3 3 2 2 1 Probit mortality Probit
Probit mortality Probit 1 0 0 0.5 0.7 0.9 1.1 1.3 1.5 0.5 0.7 0.9 1.1 1.3 1.5 Log dose Log dose
3 hrs exposure 3 hrs exposure 6 6 5 5 4 4 3 3 2 2 1 1 Probit mortality Probit Probit mortality Probit 0 0 0.5 0.7 0.9 1.1 1.3 1.5 0.5 0.7 0.9 1.1 1.3 1.5 Log dose Log dose
6 hrs exposure
6 hrs exposure 6 8 5 6 4 3 4 2 2
Probit mortality Probit 1 Probit mortality 0 0 0.5 0.7 0.9 1.1 1.3 1.5 0.5 0.7 0.9 1.1 1.3 1.5 Log dose Log dose
12 hrs exposure 12 hrs exposour 7 6
y 6 5 5 4 4 3 3 2 2
Probitmortalit 1
Probit mortality Probit 1 0 0 0.5 0.7 0.9 1.1 1.3 1.5 0.5 0.7 0.9 1.1 1.3 1.5 Log dose Log dose
Trix Vim
Fig. 1. Contact toxicity (LC50) of Trix and Vim against adult P. americana (N=12) at different exposure periods. Toxicity of dishwashing liquids against the cockroach 55
Conclusion Cornwell, P.B. 1968. The Cockroaches, Vol. 1. Hatchinson, London. Cockroaches have developed resistance against most of the chemical insecticides, but the mode of Moore, W.S., Profita, J.C. & Kochler, C.S. 1979. Soaps action of liquid detergents possibly would for home landscape insect control. Calif. Agric. 33:13-14. overcome this problem. Further studies with liquid detergents are needed to establish the range of Szumlas, D.E. 2002. Behavioural responses and lethal concentrations against the egg and nymph mortality in German Cockroaches (Blattodea: stages of cockroaches. From the results of the Blattellidae) after exposure to dishwashing liquid. present study it can be concluded that the J. Econ. Entomol. 75(2):390-398. commercial liquid detergents can effectively be Ware, G.W. 2000. The pesticide book, 5th edn. used in cockroach control. Thamson Fresno, CA. References Abbasi, S.A., Nipaney, P.C. & Soni, R. 1984. Soap solution as an environmentally safe pesticide for household insects a preliminary investigation. Comp. Physiol. Ecol. 9:46-48.
Manuscript received on 06.02.2011, Accepted on 18.04.2011 Univ.Determination j. zool. Rajshahi. of immune Univ. responseVol. 29, 2010 in quailpp. 57-60 ISSN 1023-61041 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Determination of immune response against alum-precipitated fowl cholera vaccine in the quail, Coturnix japonica M.T. Iqbal1, M.H. Haque*, S. Sarker2, M.A. Islam1 and K.A. Choudhury1 1Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh, 2Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi-6205, Bangladesh, *Corresponding Author: DR. Md. Hakimul Haque, Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi-6205, Bangladesh; E-mail: [email protected]
Abstract: The immune response of a formalin-inactivated alum-precipitated fowl cholera vaccine (FCV) was evaluated in quails, Coturnix japonica (Order: Galliformes and Family: Mimidae). All quails, irrespective of sex and 8-weeks-old were immunized with 5×107CFU/ml/quail subcutaneously (SC) and intramuscularly (IM). A Booster immunization was given with similar dose and routes at 15 days after primary immunization in groups A and B while group C served as unimmunized control. Pre-immunized sera were collected from all groups of birds to assay the primary antibody levels in them. Sera of the immunized and control quails were collected at 15 and 30 days post immunization. The degree of immunity produced in each group of quails following primary and secondary immunizations were determined by measuring their serum antibody titres using passive haemagglutination assay (PHA) test. The level of antibody was significantly increased both primary and booster immunization in immunized quails as determined by PHA titres. Two weeks after final immunization, the quails were challenged with a virulent isolate of fowl cholera and immunized quails conferred 100% protection while all the control quails were dead within 10 days post challenge.
Key words: Fowl cholera, alum-precipitated vaccine, immune response, PHA test, quail
Introduction vaccines (Khan et al., 1994; Islam et al., 2004) and comparative efficacy of different FCV Fowl cholera (FC) is one of the most important (Choudhury et al., 1988) have been evaluated contagious bacterial diseases of poultry caused under local conditions. But vaccination has not yet by the bacterium Pasteurella multocida. It occurs been practiced in quail against FC. Quail is sporadically or enzootically in most countries of reported to be more resistant to many infections the world including Bangladesh (Heddleston & as reported earlier (Heddleston & Rhoades, 1978) Rhoades, 1978; Choudhury et al., 1985). Most but presently there are many unpublished reports reported outbreaks of FC are in chickens, turkeys, from farmers of the incidence of FC like infection ducks, geese and also in quails (Heddleston and in quail. Although various studies have been Watko, 1965; Choudhury et al., 1985). FC conducted on the FC in experiment models (Islam typically occurs as a fulminating disease with et al., 2004), but there is no available information massive bacteraemia and high morbidity and regarding the immune responses against FC mortality. Vaccination is practiced as preventive vaccine in this bird. The present research measures in Bangladesh like in other countries of therefore was conducted to determine the efficacy the world to reduce the incidence of the disease and safety level of alum-precipitated FCV with (Choudhury et al., 1988). Both humoral and cell their immunogenic response in quail. mediated immunity are considered to be of primary importance in the protection of animals Materials and Methods and birds against infectious diseases (Collins, A total of 30 eight-week-old quails Coturnix 1977; Mondal et al., 1988). The immune japonica (Galliformes:Mimidae) used for this study responses (Mondal et al., 1988; Choudhury et al., were purchased from the Bangladesh Agricultural 1990), efficacy of oil adjuvanted broth culture University (BAU) poultry farm, Mymensingh. (Choudhury et al. 1987), alum-precipitated 58 Iqbal et al.
These quails were reared in the poultry shed of (1988) whereas the mean antibody titres were the Department of Microbiology and Hygiene, 102.4±33.05 in both groups of quails after booster BAU, Mymensingh supplying food and water ad immunization which corresponds with the findings libidum with maintaining proper biosecurity. of Choudhury et al. (1987) and Mondal et al. Pasteurella multocida (PM-38) serotype 1(X-73) (1988). The mean pre-vaccination PHA titre of was collected from the same Department and sera samples of all immunized and control quails used for the preparation of formalin killed alum- were found to be 4±0, which correlated with precipitated FCV as well as challenge virulent finding of Mondal et al. (1988) (Fig. 1). organisms. The quails were equally divided into three experimental groups namely A, B, and C 250 Gr B and were maintained in separate cages. Each of Gr A the quails of A and B groups was immunized with 200 7 0.5 ml of 5×10 CFU of the FCV through 150 subcutaneous (SC) and intramuscular (IM) route, respectively. Booster immunization was 100 PHA titres administered in A and B groups after 15 days of 50 primary immunization. Quails of the group C served as unimmunized control throughout the 0 experimental period. 60 days 75 days 90 days Days The immune response was studied by using passive haemagglutination assay (PHA) and protection tests to determine the presence of Fig. 1 Comparison of serum PHA titres of quails antibody against P. multocida in the serum of vaccinated with fowl cholera vaccine quails immunized with the prepared FCV. PHA through SC in Gr A and IM in Gr B test was conducted according to the procedure Protection test: Quails of groups A and B showed described by Tripathy et al. (1970) and Islam et al. 100% protection against challenge infection (2004). The protection test was conducted on both whereas quails of the group C showed 0% immunized and control groups of quails with the protection (Fig. 2). Immunized quails protected same dose rate mentioned above through IM challenge infection showed no clinical signs route as described by Choudhury et al. (1985) and except for dullness, depression and drowsiness. Islam et al. (2004). The challenged quails were In controls, the clinical signs first observed at 12 observed up to 10 days for the development of hours post inoculation (PI) were characterized by any clinical signs and symptoms of FC. P. dullness and depression. There were dullness, multocida was reisolated from challenged quails depression, slight rise of body temperature according to the method of Choudhury et al. ° (42.5 C) and increased respiratory rates (30- (1987). Statistical significance was evaluated 45/min) at 24 hours PI. At 48 hours PI, severe using student’ t test. All the analyses were weakness, drowsiness, anorexia, rise of body performed using SPSS (10.0 version). A p-value ° temperature (43.6 C), increased respiratory rates of <0.05 being considered as statistically (45-55/min), lameness and whitish (chalky) significant. diarrhoea with mucous were the important clinical Results and Discussion manifestations which was previously observed by many authors’ (Sharma et al. 1974; Gordon and PHA test: The test was conducted to determine Jordan, 1985; Rhoades and Rimler, 1990). The the immune response induced in quails having clinical signs at 96 hours PI were almost similar to been inoculated to 8-week-old quails with the that of 48 hours. Death of one quail was observed antigen containing P. multocida. In this respect, first at 24 hours PI, while two died at 48 hours and Choudhury et al. (1985); Chang (1987); Mondal et others died at 96 hours PI. al. (1988) and Sarker et al. (1992) stated that PHA test was useful for assay the serum antibody titres Post challenge isolation of bacteria: P. multocida following administration of FC vaccine. The mean were reisolated from heart, blood, liver and brain antibody titres of quails of group A and B were of quails of all groups. 51.2±16.52 and 57.6±29.4, respectively after Independent samples t-tests revealed that primary immunization which is similar to the immune response was non significant due to route findings of Coates et al. (1977) and Mondal et al. variation. Paired samples t-tests showed that Determination of immune response in quail 59
immune response of secondary immunization was Choudhury, K.A., Mondal, S.K., Rahman, M.M., Amin, significantly higher (102.4) than the primary M.M. & Sarkar, A.J. 1990. Changes in leukocytes, immunization. The mean PHA titre after primary total serum protein and immunoglobulin levels in and secondary immunization induce better chickens immunized against fowl cholera. The Bang. Vet. 7(1):27-30. immune response in quail but no remarkable variation in immune response were recorded Coates, R.S., Jensen, J.M. & Brown, E.D. 1977. The between two routes of vaccination. Thus it may be response of turkey to varying doses of lives oral concluded that the route did not cause any Pasteurella multocida vaccine. Inter. J. Poult. Sci. variation in production of immune response. The 56:273-276. present finding did not correlate with many of the Collins, F.M. 1977. Mechanisms of acquired resistance investigators. The experimental FCV conferred of Pasteurella. Cornell Vet. 67:103-167. 100% protection (P<0.01) against challenge Gordon, R.F. & Jordan, F.T. (eds.). 1985. Poultry infections. Diseases. (2nd edn.), ELBS, Baillier Tindall, London, pp. 44-46. 100 Heddleston, K.L. & Rhoades, K.R. 1978. Avian
) 80 pasteurellosis in diseases of poultry. (7th edn.),
%
(
e Iowa State University Press, Ames. Iowa, USA, pp.
t
a
r 60 181-199.
y
t
i Survivality rate (%)
l a 40 v Heddleston, K.L. & Watko, L.P. 1965. Fowl cholera:
i
v r comparison of serologic and immunogenic
u S 20 responses of chicken and turkeys. Avian Dis. 0 9:367-376. ABC Groups Islam, M.A., Samad, M.A. & Rahman, M.B. 2004. Evaluation of alum precipitated formalin killed fowl Fig. 2. Survivality of quails immunized with cholera vaccines with their immunologic responses formalin killed allum precipitated FCV. in ducks. Inter. J. Poult. Sci. 3:140-143. Therefore, it may be suggested the formalin killed Khan, M.A.H.N.A., Das, P.M., Choudhury, K.A. & Islam, alum-precipitated FCV prepared with highly M.R. 1994. Pathology of experimentally induced fowl cholera in chickens. Bang. Vet. J. 31(1-2):28- antigenic strain of P. multocida should be used to 34. provide better protection against the epidemic FC in quail. However, further study with large number Mondal, S.K., Choudhury, K.A., Amin, M.M., Rahman, of quails to determine the efficacy of routes of M.M. & Sarker, A.J. 1988. Immune response in immunization and establishment of Enzyme linked chickens induced by Alum precipitated fowl cholera vaccine. L Humoral immune response. Bang. Vet. immunosorbent assay test instead of PHA for J. 22:7163-69. determination of immune response is necessary to conclude about the present study. Rhoades, K.R. & Rimler, R.B. 1990. Pasteurella multocida colonization and invasion in References experimentally exposed turkey poults. Avian Dis. 34(2):381-383. Chang, C.F. 1987. In vitro assay for humoral and cell mediated immunity to pasteurellosis in ducks. J. Sarker, A.J., Amin, M.M. & Hossain, W.M.A. 1992. Chi. Soc. Vet. Sci. 13(1):67-74. Testing and quality control of poultry vaccines and its monitoring in the field. Bang. Agri. Uni. Res. Pro. Choudhury, K.A., Amin, M.M., Rahman, A. & Ali. M.R. 6:249-257. 1985. Investigation of natural outbreak of fowl cholera. Bang. Vet. J. 19(1-4):49-56. Sharma, R.N., Bhalla, N.P. & Lall, J.M. 1974. Observation of experimental infection of Choudhury, K.A., Amin, M.M., Sarker A.J. & Ahamed, Pasteurella septica (avian) in domestic fowl. Indi. J. A.R. 1987. Immunization of chickens against fowl Ani. Heal. 13(2):105-109. cholera with oil adjuvanted broth culture vaccine. Bang. Vet. J. 21(1-2):63-73. Tripathy, D.N., Hanson, L.E. & Myers, W.L. 1970. Passive haemagglutination test with fowl pox virus. Choudhury, K.A., Amin, M.M., Sarker, A.J. & Ali, M.I.L. Avian Dis. 14:29-38. 1988. Studies on the comparative efficacy of fowl cholera vaccines. Bang. Vet. J. 22(1-2):101-108.
Manuscript received on 04.01.2011, Accepted on 14.03.2011 Univ.Rapid j. detectionzool. Rajshahi. of ILT Univ. virus Vol. by 29, PCR 2010 pp. 61-64 ISSN 1023-61041 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Rapid detection of infectious laryngotracheitis virus by standardization of polymerase chain reaction targeting a relatively conserved region of the thymidine kinase gene S. Chakma1, S. Sarker*, S. Talukder2, M.H. Haque3, E.H. Chowdhury1 and A.S.M. Bari1 1Department of Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh, 2Department of Animal Science and Nutrition, Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong-4202, Bangladesh, 3Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi-6205, Bangladesh. *Corresponding author: DR. Subir Sarker, Department of Animal Husbandry and Veterinary Science, University of Rajshahi, Rajshahi-6205, Bangladesh; E-mail: [email protected]
Abstract: Infectious laryngotracheitis is an acute upper respiratory tract infection of chickens caused by infectious laryngotracheitis virus. The study was conducted to standardize the polymerase chain reaction targeting a relatively conserved region of the thymidine kinase gene for the rapid detection of infectious laryngotracheitis virus. The vaccine samples were collected from two renowned company of Bangladesh. DNA was extracted from diluted vaccine samples by using Wizard® Genomic DNA purification kit and thymidine kinase gene was amplified by using PCR system 9600 Thermocycler. Two vaccine samples were positively amplified by polymerase chain reaction. A procedure was developed for rapid detection of infectious laryngotracheitis virus by polymerase chain reaction of the conserved region of viral thymidine kinase gene containing DNA fragments. The results obtained in this study suggested that the polymerase chain reaction procedure could serve as a fast and sensitive method for the detection of vaccine strains of infectious laryngotracheitis viruses.
Key words: Infectious laryngotracheitis virus, viral thymidine kinase (TK) gene, polymerase chain reaction
Introduction evidence that vaccine derived strains have become established in the field (Graham et al., Infectious laryngotracheitis (ILT) is a worldwide 2000). This ability of ILTV vaccine strains to re- distributed upper respiratory disease of chickens circulate may also be responsible for some (Guy & Bagust, 2003) and included within the list E outbreaks in susceptible birds, as passage in of the Office International Des Epizooties (OIE) birds has been reported to result in increasing (Hidalgo, 2003). Chicken is the only significant virulence (Guy et al., 1991), while stress in latently primary host species for infectious laryngotracheitis infected birds has also been demonstrated to be virus (ILTV) and no other reservoir species have responsible for the re-excretion of ILTV (Bagust been recognized, even though pheasant and et al., 2000). peafowl can sometimes be naturally infected by contact with chickens actively shedding ILTV (Guy & Conventional laboratory diagnosis of ILT is based Bagust, 2003). Infection is acquired via the upper on virus isolation in specific pathogen free chicken respiratory tract and transmission occurs most embryos inoculated via the chorioallantoic route or readily from acutely infected birds but clinically in primary chicken embryo kidney cells, chicken inapparent infection can persist for long periods with embryo liver cell (Tripathy, 1998). However, it is intermittent re-excretion of the virus, and these time consuming and labor intensive in spite of its recovered carrier birds are also a potential means of high sensitivity and specificity. The use of direct transmission of the disease (OIE, 2008). The fluorescent-labeled polyclonal antibodies (FA) to disease is common in areas of intense poultry detect viral antigens in tracheal and conjunctival production and causes great economic losses due smears (Goodwin et al., 1991), or monoclonal to moderate to severe mortality, and drop in egg antibodies to detect viral antigens in frozen production (Callison et al., 2007). tracheal sections have been successfully utilized in the rapid diagnosis of the disease, although Strains of infectious laryngotracheitis virus may those had comparatively poor sensitivity. vary considerably in their virulence and there was 62 Chakma et al.
Therefore, a requirement for the development of University, Mymensingh. The vaccine was diluted rapid and sensitive diagnostic techniques for the with the given sterile buffered and colored solvent verification of clinical diagnosis of ILTV for the and stored at 2-80C. improvement of the quality of surveillance DNA extraction systems. As such, the present study was carried out to standardize the polymerase chain reaction DNA was extracted from diluted vaccine samples (PCR) targeting a relatively conserved region of by using Wizard® Genomic DNA purification kit the thymidine kinase (TK) gene for the rapid (Promegra Corporation. 2800 Woods Hollow detection of ILT virus. Road. Madison, USA). The extracted DNA was quantified using a spectrophotometer’s Materials and Methods (Spectronic® GeneticsTM New York, USA) and Collection of samples for DNA extraction expressed in ng/µl. The Nobilis® ILT and Gallivac LT freeze dried Selection and synthesis of primers vaccine samples were collected respectively from An appropriate primer sequence for PCR was the Intervet, Bengal Overseas Limited and selected to amplify thymidine kinase (TK) gene Advance Animal Science Company Limited, (Griffin & Boursnell, 1990) of ILT virus. The primer Dhaka, Bangladesh, and carried in an ice box to sequences (Table 1) were synthesized the Department of Pathology, Faculty of commercially by Sangamo Biosciences, Inc., Veterinary Science, Bangladesh Agricultural Singapore.
Table 1. Primers used in PCR for infectious laryngotracheitis detection Target Product Primer Sequence Location Gene Size The ILTVPCR5' primer starts at ILTV base 222 in the keeler sequence 5'-ACGATGACTCCGACTTTC-3' PCR5' and base 4501 in the Griffin TK gene 647bp sequence. The ILTPCR3' primer starts at base 847 in the keeler ILTV 5'-CGTTGGAGGTAGGTGGTA-3' sequence and base 5130 in the PCR3' Griffin sequence.
Amplification of DNA by polymerase chain Agarose gel electrophoresis reaction (PCR) The amplified PCR products were separated by PCR amplification was performed in a final electrophoresis on 1.5% agarose gel containing volume of 10μl containing 2μl of extracted DNA 5μl ml-1 ethidium bromide with a 100 bp ladder template, Taq DNA polymerase buffer (10x) 1μl, (Promega, Madison, WI, USA) as molecular dNTPs 1μl, primer-F (50n mol) 0.5μl, primer-R weight marker (Oliveira et al., 2003). (50n mol) 0.5μl, Taq DNA polymerase 0.2μl and Documentation of the PCR products 4.8μl nuclease free water (Science Park Rd.#01- 23, The Geni S’pore). 2µl water was added After electrophoresis, the gel was taken out instead of DNA to the water control tube. carefully from the gel chamber and placed on the Amplification was carried out in Gene UV Transilluminator in the dark chamber of the amplification PCR system 9600 Thermocycler Image Documentation System (Labortechnik, (eppendorf, Germany), using condition modified Germany). from Abbas et al., (1996). The pre-mix was then mixed well through spinning. Initial denaturation Results and Discussion was at 94°C for 1 min, again denaturation was at Polymerase chain reaction was optimized and 95°C for 1 minute, annealing at 50°C for 1 minute evaluated by the amplification with primers made and extension at 72°C for 1.5 minutes, with a final from targeted portion of thymidine kinase (TK) extension at 72°C for 5 minutes for total 35 cycles gene of the ILTV. The PCR was performed at and held for 4°C in refrigerator until different annealing temperatures and products of electrophoresis. 647-bp were successfully amplified at 500C Rapid detection of ILT virus by PCR 63
annealing temperature. The expected fragment of In spite of the use of vaccines for the control of 647-bp of the thymidine kinase gene of ILTV was ILTV, the disease continues to be a problem in obtained from two samples, as well as no band commercial poultry (Clavijo & Nagy, 1997). The was detected in the negative control (Fig 1). The use of live and modified live vaccines has been positive result in the PCR directed to the demonstrated to lead to spread of the virus, thymidine kinase gene of infectious particularly within 7-10 days of vaccination. laryngotracheitis virus (ILTV) agrees with the Carriers may result, and it is possible that the findings of Abbas et al. (1996). infection become indigenous and thus the vaccine strains of the virus may be involved in ILTV
C M S1 S2 outbreaks (Hughes et al., 1987). These findings provide support for the requirement of continuous monitoring of the vaccine strains and the development of molecular techniques that may allow simple and reliable identification of different vaccine strains. Acknowledgements The authors would like to express the deepest 647-bp sense of gratitude to the Department of Pathology, Bangladesh Agricultural University, Mymensingh for providing laboratory facilities with necessary research materials.
References Abbas, F., Andreasen, J.R. & Jackwood, M.W. 1996. Development of a Polymerase chain reaction and a nonradioactive DNA probe for infectious laryngotracheitis virus. Avian Dis. 40:56-62. Bagust, T.J., Jones, R.C. & Guy, J.S. 2000. Avian Fig 1. Visualization of the 647-bp PCR product from the infectious laryngotracheitis. Rev. Sci. Tech. 19:483- thymidine kinase gene of ILTV by agarose gel 492. electrophoresis (1.5%) after staining with ethidium ® bromide. Lane-S1: viral vaccine (Nobilis ILT); Lane-S2: Callison, S.A., Riblet, S.M., Oldoni, I., Sun, S., Zavala, viral vaccine (Gallivac LT); Lane-M: molecular marker; G., Williams, S., Resurreccion, R.S., Spackman, E. Lane -C: negative control & Garcia, M. 2007. Development and validation of Molecular based diagnostic methodologies, such a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus as DNA probes (Key et al., 1994) and the in poultry. J. Virol. Metho. 139:31-38. polymerase chain reaction (Williams et al., 1994; Abbas et al., 1996) have been developed to Clavijo, A. & Nagy, E. 1997. Differentiation of infectious diagnose the infectious laryngotracheitis virus. laryngotracheitis virus strains by polymerase chain The positive results in the PCR directed to the reaction. Avian Dis. 41:241-246. thymidine kinase gene of ILTV is a useful tool to Forghani, B. & Erdman, D.D. 1994. Amplification and confirm the diagnosis of birds suspected to detection of viral nucleic acids. In: Schmidt JN, infectious laryngotracheitis. Results can be Emmons RW. Diagnostic procedures for viral, obtained in less than 24 hours which is an rickettsial and chlamydial infections. Washington, essential point in outbreaks when fast decisions DC: American Public Health Association. pp. 97- 120. are required. The application of the procedures described herein must be evaluated for field Goodwin, M.A., Smeltzer, M.A., Brown, J., sample collected from suspected birds Resurreccion, R.S. & Dickson, T.G. 1991. considering that PCR applied to viral diagnosis is Comparison of histopathology to the direct a highly sensitive technique (Forghani & Erdman, immunofluorescent antibody test for the diagnosis of infectious laryngotracheitis in chickens. Avian 1994) that allows the detection of infection in a Dis. 35:389-391. very early phase when compared to serological reactions (Pang et al., 2002). 64 Chakma et al.
Graham, D.A., McLaren, T.E., Calvert, V.M., Torrens, Oliveira, S.D., Roclenbuisch, C.R., Ce, M.C., D. & Meehan, B.M. 2000. RFLP analysis of recent Rocha, S.L.S. & Canal, C.W. 2003. Evaluation of Northern Ireland isolates of infectious selective and non- selective enriched PCR laryngotracheitis virus: Comparison with vaccine procedures for Salmonella detection. Letters in virus and field isolates from England, Scotland and Application Microbiology. 36:217-221. the Republic of Ireland. Avian Pathol. 29:57-62. Pang, Y., Wang, H., Girshick, T., Xie, Z. & Khan, M. Griffin, A.M. & Boursnell, M.E.G. 1990. Analysis of the 2002. Development and application of a multiplex nucleotide sequence of DNA from the region of the polymerase chain reaction for avian respiratory thymidine kinase gene of infectious agents. Avian Dis. 46:691-699. laryngotracheitis virus; potential evolutionary relationships between the herpesvirus subfamilies. Tripathy, D.N. 1998. Infectious laryngotracheitis. In: J. Gen. Virol. 71:841-850. Swayne, D. E., Glisson, J. R., Jackwood, M. W., Pearson, J. E., Reed, W. M. (Eds.), A laboratory Guy, J.S. & Bagust, T.J. 2003. Laringotracheitis. In: Saif Manual for the isolation and identification of Avian Y.M., Barnes, H. J., Fadly, A. M., Glisson, J. R., pathogens. American Association of Avian McDougald L. R. and Swayne, D. E. (eds.) Pathologist, Kennett Square, PA, pp. 111-115. Diseases of poultry (11th edn.). pp. 121-134. Iowa State University Press, Amer. IA. Williams, R.A., Savage, C.E. & Jones, R.C. 1994. A comparison of direct electron microscopy, virus Guy, J.S., Barnes, H.J. & Smith, L.G. 1991. Increased isolation and a DNA amplification method for the virulence of modified-live infectious detection of avian infectious laryngotracheitis virus laryngotracheitis vaccine virus following bird-to-bird in field material. Avian Pathol. 23:709-720. passage. Avian Dis. 35:348-355. Hidalgo, H. 2003. Infectious laryngotracheitis: A Review. Brazilian J. of Poul. Sci. 5: 157-168. Hughes, C.S., Jones, R.C., Gaskel, R.M., Jordan, F.T.W. & Bradbury, J.M. 1987. Demonstration in live chickens of the carrier state in infectious laryngotracheitis. Res. Vet. Sci. 42:407-410. Key, D.W., Gough, B.C., Derbyshire, J.B. & Nagy, E. 1994. Development and evaluation of a non- isotopically labelled DNA probe for the diagnosis of infectious laryngotracheitis. Avian Dis. 38:467-474. OIE, 2008. OIE Terrestrial Manual. Chapter 2.3.3 Avian infectious laryngotracheitis.
Manuscript received on 04.01.2011, Accepted on 23.02.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 65-68 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Genetic variation of some quantitative traits in ten exotic varieties of silkworm, Bombyx mori L. Md. Kamrul Ahsan and S.M. Rahman Department of Zoology, University of Rajshahi, Rajshahi-6205, Rajshahi.
Abstract: An investigation was conducted with ten exotic varieties of silkworm, Bombyx mori L. to estimate variability, heritability and genetic advance for nine quantitative characters. A wide range of variation was observed for the majority of the traits and a major portion of the total phenotypic variation was of genetic in nature. The maximum phenotypic and genotypic coefficient of variation was found in filament length (21.45, 21.44) and the minimum in hatching percentage (1.52, 1.44), respectively. A moderate to high heritability was recorded for these varieties. Filament length showed the highest heritability (99.98). A comparatively high heritability accompanied by a high genetic advance was also noted for majority of the traits. Substantial variability, high heritability and high genetic advance for filament length, weight of mature larvae, total number of eggs laid per female and effective rate of rearing by weight recorded in this study would afford scope for effective selection.
Key words: Bombyx mori, genetic variation, exotic varieties.
Introduction selected from Bangladesh Sericulture Research and Training Institute, Rajshahi constituted the Collection of varietal materials of mulberry base material of this experiment. Standard rearing silkworm, Bombyx mori L. and their evaluation is a techniques recommended by Krishnaswami pre-requisite for the improvement of the economic (1978) were followed. Disease free layings (dfls) traits (Akanda et al., 1998, Ahsan & Rahman, from each breed were reared on rearing trays in a 2008). But very little attention has so far been randomized block design with three replications. given for the improvement of mulberry silkworm in Nine quantitative characters such as total number Bangladesh. The detailed genetic analysis of of eggs laid per female (TEL), hatching quantitative characters of the available varieties of percentage (HP), weight of mature larvae (LW), B. mori has not been investigated till to date effective rate of rearing by number (ERRn), although studies on the variation of yield and yield effective rate of rearing by weight (ERRw), cocoon contribution characters are of great importance for weight (CW), shell weight (SW), filament length the improvement of silk yield (Rahman & (FL) and estimated cocoon yield per 100 dfls Khalequzzaman, 1993; Reza & Rahman, 1996, (ECY) were collected from each replication for 2005). The progress and success in any breeding 2 statistical analysis. Genotypic (δ g) and programme for evolving superior breeds depends 2 phenotypic (δ p) variations were estimated by the upon the nature and magnitude of genetic formula given by Burton & De Vane (1953) and variability in the available materials. If the the coefficient of variability was worked out as variability in the population of largely in genetic described by Burton (1952). Heritability in broad nature with least environmental effect, the sense was estimated by the following formula probability of isolating superior genotype is high. given by Hanson et al. (1956). Since this variability is highly influenced by the environment, it becomes difficult to predict the Heritability (Broad sense) = δ 2g/ δ 2p x 100 contribution of genetic factors against The expected genetic advance was estimated by environmental effects. With this back ground, the the following formula: present investigation was undertaken to study the genetic variability among ten exotic stocks 2 2 2 available in Bangladesh. Genetic advance (GA) = δ g / δ p x k δ p. Materials and methods Where k has a value of 2.06 which is the expectation in 5% selection intensity from a large Ten exotic varieties of silkworm namely, Dong - population (Lush, 1949; Miller et al., 1958) 34(M), Ziangsu - 12(J), RB - 001, RB - 112, Bivoltine - J, NB7, NB18, NB4D4, SK1 and SK2 66 Ahsan & Rahman
Results and Discussion (5.38) and lowest in shell weight (0.15). The genotypic coefficient of variation alone is not Analyses of variances of nine quantitative sufficient to determine the amount of variation characters are shown in Table1. The genotype which is heritable. The heritable portion of items showed highly significant results indicating variation can be found from heritability estimates pronounced racial diversity have been and genetic gain (Johnson et al., 1955). In this substantiated by the finding of Tayade 1987, study the highest heritability (Hb) was obtained for Ahsan et al., 2001; Reza & Rahman, 2005. FL (99.89) followed by LW, TEL, FRRw, CW, HP, Similar results on varietal differences with respect FRRn, SW and lowest in ECY (72.29). The to larval and cocoon characters in B. mori have genetic advance (GA) estimated for different been reported by Sidhu et al. (1969), Ahsan et al. characters ranged from 0.087 (SW) - 363.45 (FL). (1998) and to larval characters by Reza et al. When genetic advance was expressed as (1993). The replication items revealed insignificant percentage of respective means (GA%), highest results showing homogeneity in different parts of score was noted in FL (44.15) followed by SW, the present investigation. LW, TEL, ERRw, CW, ECY, ERRn and lowest in HP(2.82). The results on overall range, mean with SE and genetic variability for different characters are In this results FL, LW, TEL and ERRw showed shown in Table 2. Phenotypic variances (δ 2p) comparatively high coefficient of variability, were generally greater than their corresponding heritability, as well as genetic advance and genotype (δ 2g) or environmental (δ 2e) variances genetic advance as percentage of mean indicates in all the cases. Closer δ 2p and δ 2g as well as that a wide range of genetic diversity existed higher δ 2g than δ 2e for all the characters which could be used in a breeding programme. It indicated that the major part of the phenotypic also indicated the importance of additive gene variation was of genotypic in nature. effects in these characters and phenotypic selection of these characters would be effective Estimates on phenotypic (CVp), genotypic (CVg) (Panse, 1957; Rahman & Rahman, 1990; Reza and environmental (CVe) co-efficient of variability, et al., 1993). heritability (Hb), genetic advance (GA) and genetic advance as percentage of mean (GA%) have A number of characters showed high heritability been presented in Table3. The maximum but low genetic advance. This might happen due phenotypic and genotypic coefficient of variation to the intra or inter allelic interaction. In such was found in filament length and the minimum in situation recurrent selection may be adopted to hatching percentage. The range of phenotypic co- these trails (Pershad et al., 1986, Rahman & efficient of variation was from 1.52 - 21.45 and of Rahman, 1990; Ahsan & Rahman 2008). genotypic was 1.44 - 21.44. Where as the highest environmental coefficient of variation was recorded in estimated cocoon yield / 100 dfls
Table 1. Mean square of ANOVA for nine quantitative characters in B. mori. MS df TEL HP LW ERRn ERRw CW SW FL ECY Variety 9 6943.63** 5.35** 1.47** 6.48** 182.24** 0.0422** 0.00656** 93521.05** 17.38** Replication 2 14.09 0.02 0.0039 0.025 0.67 0.0002 0.00049 24.62 2.73 Error 8 18.64 0.19 0.0037 0.27 .097 0.00037 0.00035 34.56 1.97 ** p < 0.001 Table 2. Range, Mean with SE and components of variance of different characters in B. mori. Characters Range Mean SE δ 2p δ 2g δ 2e TEL 445.17 - 70.18 518.35 3.53 2326.97 2308.33 18.64 HP 88.50 - 94.00 91.02 0.35 1.91 1.72 0.19 LW 3.25 - 5.00 4.16 0.05 0.4925 0.4888 0.0037 ERRn 64.00 - 69.51 66.25 0.42 2.34 2.07 0.27 ERRw 76.52 - 97.40 87.76 0.50 61.39 60.42 0.97 CW 1.14 - 1.46 1.33 0.0157 0.01431 0.01394 0.00037 SW 0.17 - 0.29 0.233 0.0153 0.00242 0.00207 0.00035 FL 605.17- 1000 823.28 4.8 31196.72 31162.16 34.56 ECY 19.28 - 29.58 26.08 1.15 7.11 5.14 1.97 Genetic variation of quantitative traits of silkworm 67
Table 3. Phenotypic (CVP), genotypic (CVg) and environmental (CVe) coefficient of variation, heritability (Hb), genetic advance (GA) and genetic advance as percentage of means (GA%) of different characters in B. mori. Characters CVp CVg CVc Hb GA GA% TEL 9.31 9.27 0.83 99.20 98.58 19.02 HP 1.52 1.44 0.48 90.05 2.54 2.82 LW 16.87 16.81 1.46 99.25 1.43 34.49 ERRn 2.31 2.17 0.78 88.46 2.79 4.21 ERRw 8.93 8.85 1.12 98.42 15.89 18.10 CW 8.99 8.88 1.45 97.41 0.24 18.05 SW 21.11 19.53 0.15 85.54 0.087 37.20 FL 21.45 21.44 0.71 99.89 363.45 44.15 ECY 10.22 8.69 5.38 72.29 3.97 15.25
References Panse, V.G. 1957. Statistical methods for agricultural works. I.C.A.R., New Delhi..241 pp. Ahsan, M.K. & Rahman, S.M. 2008. Genetic variability and correlation analysis in hybrids of silkworm, Pershad, G.D., Datta, R.K., Bhargave, S.K., Bombyx mori L. for egg characters. Univ. j. zoo/. Vijayakumar, H.V. & Jolly, M.S. 1986. Rajshahi Univ. 27:13-16. Performance of some multivoltine races of Bombyx mori L. Sericologia. 26:295-301. Ahsan, M.K., Rahman, S.M., Haque, M.T. & Ali, I.A. 2001. Interrelationship between some egg and Rahman, M.S. & Rahman, S.M. 1990. Estimates of larval characters in different breeds of silkworm, variability and some genetic parameters in eri Bombyx mori L. Univ. J. Zoo/. Rajshahi Univ. silkworm, philosamia ricini Bosisd. Bangladesh. J. 20:81-84. Zoo/. 18(2):239-244. Ahsan, M.K., Rahman, S.M. & Ali, I.A. 1998. Inheritance Rahman, S.M & Khalequzzaman, M. 1993. Studies on of some quantitative traits in fifteen indigenous variation in egg laying and some of its varieties of silkworm, Bombyx mori L. Univ. j. zoo/. components in silkworm. B. mori 1,. Bangladesh Rajshahi Univ. 18:79-83. J. Zoo/. 21(1):95-101. Akanda, M.A.L., Alam, M.S. & Uddin, M.M. 1998. Reza, A.M.S. & Rahman, S.M. 1996. The genetic Genetic variability, correlation and path analysis in variability, heritability and genetic advance in composite maize. Bangladesh J. Agri. Res. 23(1): silkworm, Bombyx mori L. Bangladesh. J. Agri. 107-113. 21:1-6. Burton, G.W., 1952. Quantitative inheritance in grass. Reza, A.M.S. & Rahman, S.M. 2005. Genetic Proc. 6th Inter. Grassland Cong. 1:277-283. parameters of some yield and yield contributing traits in silkworm, Bombyx mori L. Univ. j. zoo/. Burton, G.W. & De Vane, E.H. 1953. Estimating Rajshahi Univ. 24:55-58. heritability in tall fescue (Fescuta arundinaceae) from replicated clonal materials. Agron. J. 45:579- Reza. A.M.S, Rahman, M.S. & Rahman, S.M. 1993. 604. Studies on the variation of some larval traits in different breeds of silkworm, Bombyx mori L. Univ. Hanson, C.H., Robinson, H.F. & Comstock, R.E. 1956. j. zoo/. Rajshahi Univ. 12:21-24. Biometrical studies of yield in segregation populations of Korean lespedeza. Agron. 48:268- Sidhu, N.S., Singh, R.K., Pillai, V.S. & Sreenivasan, R. 272. 1969. Performance of some of the breeds of silkworm, Bombyx mori L. Silkworm Inf. Bull. 1: Johnson, H.W., Robinson, H.F. & Comstock, R.E. 1955. 41- 53 Estimates of genetic and environmental variability in soyabean. Agron. J. 47:314-318. Tayade, D.S. 1987. Performance of different races of silkworm, Bombyx mori under Marathwada Krishnaswami S. 1978. New Technology of silkworm conditions. Sericologia. 27:381-389. rearing. Central Silk Board, India. 23 pp. Lush, J.L. 1949. Heritability of quantitative characters in farm animals. Heriditas (suppl.). 35:256 -287. Miller, P.A., Williums, J.C., Robinson, H.F. & Constock, R.E. 1958. Estimates of genotypic and environmental variances and co-variances in upland cotton and their implication in selection. Agron. J. 50:126-131.
Manuscript received on 25.07.2010, Accepted on 08.04.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 69-72 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Short Note
Occurrence of protozoans & their limnological relationships in some ponds of Mathbaria, Bangladesh Pronob Kumar Mozumder1*, Marjia Akhter Banu1, M. Niamul Naser1, Md. Shahadat Ali1, Munirul Alam2, R. Bradley Sack3, Rita R. Colwell4 & Anwar Huq5 1Department of Zoology, University of Dhaka, Bangladesh ; 2Enteric & Food Microbiology Laboratory, ICDDR, B. Dhaka ; 3Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA ; 4Center for Bioinformatics & Computational Biology, University of Maryland, College Park, USA ; 5Maryland Pathogen Research Institute, University of Maryland, USA. *Corresponding author E-mail: [email protected]
Sample collection and identification of protozoans (1960), Mellanby (1975), Bhouyain & Asmat Water samples were collected fortnightly between (1992), & Ali & Chakrabarty (1992). October 2004 to May 2005 from four water bodies Protozoans composition of Mathbaria of Pirojpur district in Bangladesh. For In this study 3 genera of the protozoans were zooplankton sample collection, 100 liters of water identified from four ponds of Mathbaria. From the were filtered successively through 20 µm mesh accumulated data in the total study period nylon nets (Millipore corp., Bedford, MA), and 50 abundance of protozoans per sampling (per week) ml of the concentrates were collected initially as a varied from 15 individuals/l in March’2005 to 196 crude measure of zooplankton. From 50 ml individuals/l in October’2004 with an average of concentrates, 10 ml was used for analysis and the 93 individuals/l. (Table 1). samples were immediately preserved in 5% buffered formaldehyde solution. The bottle Variations in the percentage composition of containing the zooplankton was gently swiveled average protozoans during the study period were, (clock wise) for equal suspension of zooplankton 1.91% in Kachisira pond, 55.5% in South in the sampled water. By using a clean large Mithakhali pond, 10.53% in Mathbaria Bazar pond mouth 10 ml pipette, 1µl of suspended material and 32.06% in BRAC pond (Table 2). was placed in counting cell slot. The counting cell Variations in total protozoans during the study was then covered with a glass slide and examined period were 114 ind./l in Kachisira pond, 3305 under a compound binocular microscope at a ind./l in South Mithakhali pond, 627 ind./l in magnificent of 40 X to 100 X. The Protozoan Mathbaria Bazar pond and 1909 ind./l in BRAC samples were observed under a compound pond (Fig.1,Table 2). microscope (Axioskop 40, Japan). For protozoans From this study of four ponds in Mathbaria analysis, heliber bacteria single round cell (Cat Holophrya sp. was 0.96%, Glaucoma sp. 74.16%, no. Z 30000 LOT 11756) was used. The Nassula sp. 12.44% and unidentified protozoans specimens were identified up to genera level. were 12.44% of total protozoans in total study Quantitative analysis was followed by the total period (Table 2, Fig.2). count method of Welch (1948). Identification was made following Ward & Whipple (1959), Tonapi 70 Pronob et al.
Table 1. Pond wise variations in the abundance (Individual/l) of protozoan fauna in Mathbaria during the period of October 2004 to May 2005. Ponds Months Oct. Nov. Dec. Jun. Feb. Mar. Apr. May. Weeks Weeks Weeks Weeks Weeks Weeks Weeks Weeks Average 1st 3rd 1st 3rd 1st 3rd 1st 3rd 1st 3rd 1st 3rd 1st 3rd 1st 3rd Kachisira pond 0 57 0 0 0 0 0 0 0 0 57 0 0 0 0 0 7 South Mithakhali pond 0 741 342 285 114 114 228 342 342 455 285 0 57 0 0 0 207 Mathbaria Bazar pond 0 0 0 114 0 0 242 57 57 0 0 0 0 57 0 0 33 BRAC pond 0 0 0 0 57 114 228 57 114 0 57 57 285 542 0 455 123 Average 0 196 86 100 43 57 175 114 129 114 100 15 85 150 0 114 93
Table 2. Percentage composition of different protozoans in four ponds during the study period in Mathbaria. Ponds Unidentified Holophrya sp. Glaucoma sp. Nassula sp. Total protozoans protozoans Individual/l % Individual/l % Individual/l % Individual/l % Individual/l % Kachisira pond 57 50 0 0 0 0 57 50 114 1.91 South Mithakhali pond 0 0 2735 82.75 0 0 570 17.25 3305 55.50 Mathbaria Bazar pond 0 0 57 9.09 456 72.93 114 18.18 627 10.53 BRAC pond 0 0 1624 85.07 285 14.93 0 0 1909 32.06 Total 57 0.96 4416 74.16 741 12.44 741 12.44 5955 100 Average 14 1,104 185 185 1,489
Relationship among the different physico- chemical variables and protozoans in the ponds. The coefficient of correlation between physico- chemical parameters and protozoans of four ponds during the study period were calculated (Table 3) .The relationships are summarized below: Water temperature showed direct positive relationship with air temperature (r=0.941). Water temperature showed positive relationship with pH (r=0.676) and DO (r=0.348). pH showed positive relationship with DO (r=0.351). Fig. 1. Occurrence of different protozoan fauna in Protozoans showed positive relationship with four ponds during the study period. dissolved oxygen (DO) (r=0.227) while inversely related with water temperature (r=-0.276) and pH (r=-0.397).
Table 3. Co-efficient of correlations (r) between physico- chemical variables and protozoans composition of four ponds in Mathbaria during the study period. Sl. Parameters Co-efficient of No. correlations ‘r’ 1. Air temperature and water 0.941 temperature 2. Water temperature and pH 0.676 3. Water temperature and DO 0.348 4. pH and DO 0.351 5. Water temperature and -0.276 Fig. 2. Showing the average percentage protozoans occurrences of different groups of protozoan 6. pH and protozoans -0.397 fauna of four ponds in Mathbaria, during the study 7. DO and protozoans 0.227 period.
Larvicidal potentials of D. indica 71
Conclusion Bhuiyan, A. S. & Nessa, Q. 1988a. A qualitative study of zooplankton in relation to the physicochemical It is evident from the study that the protozoans conditions of a fish pond of Rajshahi, Univ. J. showed variation in the selected aquatic Zool. Rajshahi Univ. 17:29-31. environments. The variation depends on the Bhuiyan, A. S. & Nessa, Q. 1988b. Seasonal variation different physicho-chemical variables that exist in in the occurrence of some zooplankton in a fish the pond ecosystem. However, considering the pond. Bangladesh J. Fish. 2(2):201-203. nature of the ponds, as no fertilizer or nutrients added in the systems, natural productivity of the Dewan, S., Ali, M. & Islam, M.A.1977.Study on the size studied ponds were very high. & pattern of feeding of fries & fingerlings of three major carps, eg. Labeo rohita (Ham), Catla catla & Acknowledgements Cirrhina mrigala. Bangladesh J. Agri. 2(2):223- 228. The research is an integral part of the major multidisciplinary project entitled 'Epidemiology and Huq, A., Choudhury, M.A.R., Islam, M.S., Montilla,R. & Ecology of Vibrio cholerae in Bangladesh' was Colwell, R.R. 1996. A simple filtration method to remove plankton-associated Vibrio cholerae in raw financed by the National Institute of Health (NIH) water supplies in developing countries. Appl. research grant # 1RO1A13912901 under the Environ. Microbiol. 62(7):2508-2512. collaborative agreement between the International th Center for Diarrhoeal Disease Research, Mellanby, H. 1975. Animal Life in Freshwater. 6 ed. Bangladesh (ICDDR, B) and Johns Hopkins Trowbridge & Esher, Fedowood, Burn Ltd. pp. 308. Bloomberg School of Public Health. The authors Tonapai, G.T. 1960. Freshwater Animal in India (An gratefully acknowledge the NIH Ecological Ecological Approach). Oxford & IBH Publishing Surveillance Team at ICDDR, B for supporting this Co., New Delhi. p. 341. research. The authors are also grateful to Kaji Ward, H.B. & Whipple, C.C.1959. Freshwater Biology. Tamanna Keya, Senior Research Officer, 2nd ed. John Willy & Sons Inc., New York, London, Population Council, Dhaka, for her assistance 1248 pp. during the research. Welch, P.S. 1948. Limnological Methods. McGraw- Hill Book Co. Inc., New York. p. 381. References Ali, S. & Chakrabarty, T. 1992. Bangladesher Mitha Panir Amerudandi Prani (A book of Freshwater Invertebrates of Bangladesh). 1st edition. Bangla Academy, Dhaka, Bangladesh. p. 176. Bhouyain, A. M. & Asmat, S.M. 1992. Freshwater Zooplankton from Bangladesh. 1st ed. p.137. Bhuiyan, A. S., Islam, S. N & Bhuiyan, S.S. 2008. Seasonal occurrence of some copepods in relation to the physicochemical conditions of a fish pond in Rajshahi, Bangladesh. Fishing Chimes.India 28(4):39-41.
Manuscript received on 30.10.2010, Accepted on 21.04.2011 Univ. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 73-76 ISSN 1023-6104 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Short Note
A preliminary survey of arthropod diversity through pitfall trap in the selective habitats at Rajshahi University Campus Mosharrof Hossain Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh [email protected]
Key words: arthropod, diversity, insects, pitfall trap,
Arthropods include an incredibly diverse group of spermatophytes of grass and grass like plants of taxa such as insects, crustaceans, spiders, family Gramineae. The mustard field was chosen scorpions, and centipedes. The arthropods are for trap setting was covered with weeds. Beside essential for our existence, directly or indirectly the ponds of the RU campus, there are long providing us with food, clothing, medicines, and grassy lands shaded by medium sized bushes protection from harmful organisms. The and tall trees, and this habitat was also selected systematic relationships of arthropod groups are for trap setting. not fully understood, which is not surprising given Trap setting and Sampling: Total 36 pitfall traps the size and diversity of the phylum. were set in the three habitats. Four sticks each Arthropods are sensitive bio-indicators of one meter length was used to measure an area of environmental change because of their rapid one meter square and four traps were set in each reproductive rates, short generation times, and corner of the area. Thus three replications of the fine grain at which they occupy space in the collection site were established at each of the soil (Moldenke & Lattin, 1990). They have been three studied habitats. The traps were kept recognized as efficient indicators of ecosystem opened for 72 hours and then the mouth function and recommended for use in wasclosed and brought to the Insect Research conservation planning (Rosenberg et al., 1986; Laboratory, Department of Zoology, University of Finnamore, 1996) and many researchers have Rajshahi. The collected insects were preserved in assessed habitat quality and measured habitat 70% alcohol keeping individually in separate vials differences using arthropods (Kitchin et al., 2000; labeled marked with dates, habitats and Gibb & Hochuli, 2002). Insects are almost replication number. The traps were set every exclusively terrestrial, although a number of week for three month from December, 2003 to species are aquatic or have an aquatic stage in February 2004. The collected specimens were their life cycle, and their economic and medical recorded and identified according to Imms (1977) importance cannot be overstated. Investigation up to order level. The numbers of insects of each has been done of soil micro-arthropods in prairie order were recorded separately for each of the (Pimentel, 1961; Peters, 1997) and forest litter studied habitat. Distribution of insects at different invertebrates in tropical forest by Jansen (1997). habitats was presented by number, and that of Arthropod groups have been used to track orders found at RU campus was presented by restoration success in many contexts (Greenslade percentage. & Majer, 1993). Therefore, the present survey Total 117 specimens of different arthropods were aimed to investigate arthropod diversity and sampled using the pitfall traps set at the three density to different habitats at Rajshahi University habitats. Among 117 specimens, 60 were found in Campus (RU campus). the crop land, 37 were found in the grass lands Site selection: Three habitats, grass land, crop and 20 individuals were found beside the pond land and beside pond were selected for pitfall trap (Fig.1). Erwin (1982) reported that consistent with setting and sampling arthropods. Grass land was succession theory, arthropod diversity increased primarily composed of herbaceous with vegetation height and complexity. In this 74 Hossain
survey a similar patterns of arthropod diversity The maximum number of the total collected was found at three habitats. specimen was insects (85%) and the rest were arachnids (5%). Among the insects the highest Southwood et al. (1979) described increasing percentage was from the order Coleoptera (41%). insect diversity with increases in plants and spatial The orders Hymenoptera (26%) and Orthoptera diversity, followed by a decrease in insect (14%) were also important from the point of diversity with even higher spatial diversity but population density (Fig.2). The present survey decreasing plant diversity. They stated that there results predicting that the diversity among the is an arch in arthropod species diversity with the arthropods was also different in the three habitats. height of vegetation. In this study sites grass land Thus it is concluded that pitfall trap is easy to was more vegetated than the crop land and measure the arthropod diversity in different habitat beside pool habitat respectively. Therefore, this rapidly. finding is similar to others (Siemann, 1998; Rebek et al., 1995). This survey technique is relevant to rapid biodiversity assessment, because arthropod comprises the largest component of terrestrial biodiversity. Rebek et al., (1995) analyzed the arthropod abundance and diversity in the corn field by pitfall traps. Erwin (1982) found the highest representatives from the order Coleoptera and Hymenoptera which are similar to the present survey results. Species richness and the biological success of specific communities are positively related to the diversity of niches and soil microenvironments
(Parmelee, 1995). As a result, the extent to which cropping diversity, rotational regimes, and soil Fig. 2. Showing the abundance and diversity of preparation influence the diversity of arthropods at RU campus microenvironments in the soil tremendously impacts arthropod populations (Pankhurst, 1997). Acknowledgements Although plant diversity is thought to be overwhelmingly influential in determining regional The author is grateful to Prof. Sohrab Ali, Dean, and global arthropod diversity (Moldenke & Lattin, Faculty of Agriculture and Prof. Dr. Selina 1990; May, 1978), these results suggest it is not Parween, Department of Zoology, Rajshahi the only or perhaps not even the most important University for correction and critical suggestions to factor influencing local arthropod diversity. the manuscript. References Erwin, T.L. 1982. Tropical forests: their richness in Coleoptera and other arthropod species. Coleopterists Bull. 36:74-75. Finnamore, A.T. 1996. The advantages of using arthropods in ecosystem management. Biological Survey of Canada (Terrestrial Arthropods) for Canadian Museum of Nature and Entomological Society of Canada, Ottawa. Gibb, H. & Hochuli, D.F. 2002. Habitat fragmentation in an urban environment: large and small fragments Fig.1. Showing arthropods richness at three different support different arthropod assemblages. Bio. Con. habitats at RU campus. 106:91-100. Greenslade, P., & Majer, J.D. 1993. Recolonization by collembola of rehabilitated bauxite mines in Western Australia. Australian J. Ecol. 18:385-394. Arthropod diversity of Rajshahi University Campus 75
Imms, A.D. 1977. General and classification: General Rebek, E.J., Young, D.K. & Hogg, D.B. 1995. Textbook of Entomology, 10th ed. rev. by O.W. Pitfall trap analysis of soil macroarthropods RICHARDS and R.G. DAVIES, Vol. 2. associated with the Wisconsin Integrated Cropping Systems Trial, 1994. Fifth Report of the Jansen, A. 1997. Terrestrial invertebrate community Wisconsin Integrated Cropping Systems Trial, structure as an indicator of the success of a University of Wisconsin-Madison, pp. 52-62. tropical rainforest restoration project. Rest. Eco. 5:115-124 Rosenberg, D.M., Danks, H.V. & Luhmkuhl, D.M. 1986. Importance of insects in environmental impact Kitching, R.L., Orr, A.G., Thalib, L., Mitchell, H., assessment. Environ. Management 10:773-783. Hopkins, M.S. & Graham, A.W. 2000. Moth assemblages as indicators of environmental Siemann, E. 1998. Experimental tests of effects of plant quality in remnants of upland Australian rain forest. productivity and diversity on grassland arthropod J. Appl. Eco. 37:284-297. diversity. Ecology 79:2057-2070. May, R.M. 1978. The dynamics and diversity of insect Southwood, T.R.E., Brown, V.K. & Reader, P.M. 1979. faunas. Pages 188–204 in L. A. Mound and N. The relationships of plant and insect diversities in Waloff, eds. Blackwell, Oxford. succession. Bio. J. Linn. Soc. 12:327-348. Moldenke, A.R. & Lattin, J.D. 1990. Density and diversity of arthropods as “biological probes” of complex soil phenomena. NW Environ. J. 6:409-410. Pankhurst, C.E. 1997. Biodiversity of soil organisms as an indicator of soil health. pp. 297-324. In C. Pankhurst et al. (ed.) Biological indicators of soil health. CAB Int., New York. Parmelee, R.W., 1995. Soil fauna: linking different levels of the ecological hierarchy. In: Jones, C.J., Lawton, J.H. (Eds.), Linking Species and Ecosystems. Chapman & Hall, New York, pp. 107-116. Peters, J.R. 1997. Soil microarthropod possible indicators of prairie recovery (Illinois). Restoration & Management Notes 15:78-79. Pimentel, D. 1961. Species diversity and insect population outbreaks. Annals Entomol. Soc. America 54:76-86. U niv. j. zool. Rajshahi. Univ. Vol. 29, 2010 pp. 77-80 ISSN 1023-61041 http://journals.sfu.ca/bd/index.php/UJZRU © Rajshahi University Zoological Society
Short Note
Larvicidal effect of latex from Calotropis gigantea (L.) W.T. Aiton against the mosquito, Culex quinquefasciatus Say (Diptera: Culicidae) Khondkar Ehteshamul Kabir1, Ataur Rahman Khan2 and A.S.M. Shafiqur Rahman*2 1Laboratory of Integrated Pest Management, Institute of Biological Sciences, University of Rajshahi; 2Department of Zoology, University of Rajshahi; Rajshahi-6205, Bangladesh Key words: Culex quinquefasciatus, Calotropis gigantea, latex, larvicidal effect. *Corresponding author
The milkweed, Calotropis gigantea (L.) W.T. Aiton Test material: Milky latex of C. gigantea, grown in (Gentianales: Asclepiadaceae) locally known as the campus of Rajshahi University, Bangladesh, Akond, is an evergreen, perennial shrub, and is was collected in early-March of 2000 with widely distributed in sub-tropical and tropical punching of the young stems using sharp pins. countries. It has been reported that latices of the Before making the various concentrations of latex milkweeds including C. gigantea contain cardiac in ppm, the collected latex was poured into pre- glycosides that vary in amount and type between weighed conical flask and weighed to obtain the species, between various plant parts of the same wet weight of the latex. Then the latex was dried species and through time, and other noxious in the same flask for 24 h at 60° C, and the flasks chemical substances including pyrrolizidinic were weighed again to obtain the dry weight of the alkaloids (Farrell et al., 1991). latex. The stock solution of the semisolid latex in MeOH (w/v) was then appropriately added to the The pant latex functions as defense chemicals test water medium to obtain the desired against insect herbivores (Konno et al., 2006). concentrations for the assay. Evidence of a defensive function of cardiac glycosides in comes largely from studies of Experimental insect: All experiments and milkweed insects that sequester host plant mosquito larvae, C. quinquefasciatus Say were cardenoloides and are thus protected from reared in the laboratory of the Institute of predators. The alkaloids reported to be present in Biological Sciences, University of Rajshahi, the latex of C. procera have been shown to Bangladesh at ca. 27 ±1°C, 40-60% relative contain insecticidal properties (Larhsini et al., humidity) and a 12:12h Light: Dark (L:D) 1997). Study of Ramos et al. (2006) showed that photoperiod. The larvae were fed on brewer’s the C. procera latex contains the larvicidal yeast (Red Star®, Wisconsin, USA) at 10, 20, 40, compounds, which caused significant mortality in and 80mg/ beaker for the 1st-, 2nd-, 3rd- and 4th third stage larvae of Aedes aegypti. Public instars respectively. Rearing water was changed concern over the toxicity and environmental daily and at each time new food was added. impact of synthetic pesticides has initiated the Relative humidity is a crucial factor affecting the exploration and utilization of bioactive constituents life pattern of mosquitoes, such as mating and derived from plants to control insect pests and oviposition. vectors carrying harmful pathogens to plants and animals. Furthermore, pesticides derived from Larval bioassay procedure: Bioassays with latex natural products are less toxic to the environment on mosquito larvae were performed using a and biodegradable in nature (Isman, 2006). The standard protocol of World Health Organization aim of this study was to investigate the effect of (WHO, 1981). Twenty-five 1st-, 2nd-, 3rd- and 4th latex of C. gigantea against the larvae of the instar larvae were released into 100ml glass mosquito, C. quinquefasciatus Say, which is the beakers, containing 50ml of tap water to which the primary vector of the filarial worm, Wuchereria required volume of the latex preparation was bancrofti, causing lymphatic filariasis and of many applied, using a glass capillary micro-pipette ® different arboviruses such as WNV both in the old (WIRETROL II, Drummond Scientific Company, and new world. PA, USA) to obtain the desired concentrations, viz. 1000, 2000, 4000, 8000 and 16000 ppm. 78 Kabir et al.
Three replications were made for each Anopheles stephensi, Culex pipiens, and Aedes concentration and two controls, one with 50ml of aegypti larvae at 50000ppm (Singh, 1996). tap water alone and the other with 50ml of tap Anopheles labranchiae larvae treated with the water containing brewer’s yeast. Each replication latex water filtrate from the same species used 25 larvae. Control insects were similarly produced significant mortality at low concentration raised. (Markouk et al., 2000). The toxic effect of latex has also been reported in hoppers of the desert Statistical analysis: The mortality data were locust with the injection of latex from C. procera corrected by Abbott’s (1925) formula and (Al-Robai, 1997) and in the cabbage looper, subjected to Probit analysis according to Finney Trichoplusia ni, with the use of latex from method to estimate the concentration killing 50 Asclepias curassavica (Dussourd, 1993). The percent of the larvae tested (LC ) and their 95 th 50 present study also noted that a number of 4 percent fiducial limits (FL ). The data were further 95 instar larvae failed to ecdyse, and their outer subjected to one-way ANOVA to test the integument colour turned to black. This suggests significance between concentrations and time. that milky latex may contain phyto-ecdysteroid like In the present study the latex of C. gigantea has components, which may interfere the moulting of been used as natural bio-larvicide instead of the mosquito larvae. Besides peculiar coiling, synthetic pesticides. The results of the effect of sluggish movements were noted in treated larvae crude latex of C. gigantea on mosquito larvae at that demonstrate the effect of the latex on the various concentrations with different exposure nervous and/or muscular system. This might also periods are presented in Table 1. A perusal of the lead to subsequent mortality in the larvae. data show that the latex produced significant Curiously, first instar larvae of the monarch mortality in the mosquito larvae at different instars butterfly (Danaus plexippus L.), a milkweed compared to untreated controls and was specialist, likewise became immobilized upon moderately toxic at high concentrations. Our encountering latex of Asclepias humistrata Walt., findings found positively correlated to those of apparently due to cardiac glycosides (Zalucki & earlier works that investigated the effect of plant Brower, 1992). latex on some species of mosquitoes. Giridhar et A number of insect species from unrelated taxa al. (1984) reported that C. procera latex affects have developed adaptations to sequester mosquito larvae at high concentration. Most pyrrolizidine alkaloids and cardenolides from their recently Mwine et al. (2010) reported that host plants and to utilize them as defense Euphorbia tirucalli latex can bring about total chemicals against their own predators (Nishida, mortality of Anopheles species larvae at the 2002). The mechanism of toxic action of the highest dilution used of 1: 250 in 5 days. In our bioactive compounds in latex, especially cardiac experiment the effect of latex on larvae was found glycosides and other chemical constituents to age-and concentration-dependent. The mortalities mosquito larvae is not determined. Larval obtained at high concentrations suggest that the mortality may occur due to cardiac glycosides, activity of the bioactive compounds in the latex which inhibit the action of membrane-bound was inhibited by the influence of the + + enzyme Na /K -ATPase (Horisberger et al., 1991) microenvironment. Larvicide bioassay procedures and thus are toxic to most animals. The possible normally correlate mortality to the concentration of trade-off of mortality with high concentration material present in the medium. However, the caused by the latex requires further investigation actual dosage of test materials intended for with changing concentrations and using solvent ingestion by larvae, to serve as stomach poisons, extraction. However, the results are encouraging may be affected by various confounding factors and suggest that the crude latex of C. gigantea such as sedimentation, more or less selective could be useful as an alternative to synthetic ingestion by larvae and wide variations in the larvicides to suppress the larval populations of the larval feeding rate. Further statistical analysis of mosquito. This indigenous technology seems to the mortality data using one-way Anova detected be economically feasible for controlling the significant difference between concentrations mosquitoes in the rural regions of the South and and durations (P<0.01) (Table 2). Similar results South-East Asian countries where sophisticated have been reported with the use of latex water of and expensive methods are not available all the C. gigantea that showed 100 percent mortality in time. Larvicidal effect of C. gigantea 79
Table 1: Larvicidal effect of latex obtained from Calotropis gigantea against different larval instars of Culex quinquefasciatus.*
Larval Duration of No. of larvae LC50 values Regression equation 95% fiducial limits instar treatment (hrs) used (ppm) Lower Upper 1st 12 75 7575.47 Y= 1.29 + 0.95 X 5233.68-10965.09 24 4305.88 Y=1.74 + 0.89 X 3078.33- 6022.94 2nd 12 75 12166.47 Y= 0.96 +0.98X 7731.42-19145.61 24 8692.60 Y= 0.72 + 1.08 X 6131.73-12323.02 3rd 12 75 9374.22 Y=0.69 + 1.08 X 6527.17-13463.12 24 8672.16 Y= 0.60 + 1.11 X 6177.57-12174.12 4th 12 75 15062.6 Y= 0.20 + 1.14 X 9661.24-23483.7 24 11978.41 Y= 8.01 + 1.20 X 8261.99-17366.54 *Values were based on four concentrations, and three replications with 25 insects each
Table 2: Single factorial ANOVA for the mortality of 1st-, 2nd-, 3rd- and 4th instar larvae of Culex quinquefasciatus treated with the latex from C. gigantea. Sources of variation SS DF MS F-value Concentration *a) 1266.60 4 316.65 191.9*** *b) 1398.40 4 349.60 76.00*** *c) 1640.00 4 410.00 820.00*** *d) 1457.00 4 364.25 316.74*** Duration a) 108.90 1 108.90 66.00*** b) 19.60 1 19.60 4.26ns c) 2.50 1 2.50 5.00ns d) 14.40 1 14.40 12.52** Error a) 6.60 4 1.65 b) 18.40 4 4.60 c) 2.00 4 0.50 d) 4.60 4 1.15 Total a) 1382.10 9 b) 1436.40 9 c) 1644.50 9 d) 1476.00 9 Notes: *a= 1st instar; *b=2nd instar; *c= 3rd instar; *d= 4th instar; **P<0.05;***P<0.01; ns=not significant
Acknowledgements Dussourd, D. E. 1993. Foraging with finesse: caterpillar adaptations for circumventing plant defenses. In: The first author (KEK) would like to extend his Caterpillars: ecological and evolutionary gratitude to the Director, Institute of Biological constraints on foraging (Stamp, N. E. and Casey, Sciences, University of Rajshahi, Bangladesh, for T., eds.), Chapman and Hall, New York, pp. 92- providing laboratory facilities and fellowship, and 131. to the Ministry of Science and Technology, Farrell, B., Dussourd, D.E. & Mitter, C. 1991. Escalation Government of the People’s Republic of of plant defense: do latex/resin canals spur plant Bangladesh, for providing National Science and diversification? Am. Nat. 138:891-900. Technology Fellowship. Gatehouse, J.A. 2002. Plant resistance towards insect References herbivores: a dynamic interaction. New Phytol.156:145-169. Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. Giridhar, G., Deval, K., Mittal, P.K. & Vasudevan, P. 8:265-267. 1984. Mosquito control by Calotropis procera latex. Pesticides, 18:26-29. Al-Robai, A.A. 1997. Toxicological studies on the latex of the uscher plant Calotropis procera Ait in Saudi Horisberger, J.D., Lemas, V., Kraehenbuhl, J.P. & Arabia. IV. Effects of partly purified uscher latex Rossier, B.C. 1991. Structure-function relationship and of the poison gland secretion of the of Na, K-ATPase. Annu. Rev. Physiol. 53:565-584. uscherhopper; Poekilocerus bufonius Klug on the
desert locust, Schistocerca gregaria Forskal (Orthoptera: Acrididae). Arab Gulf J. Sci. Res. 15:709-716. 80 Kabir et al.
Isman, M. B. 2006. Botanical insecticides, deterrents, Seiber, J.N., Nelson, C.J. & Lee, S.M. 1982. and repellents in modern agriculture and an Cardenolides in the latex and leaves of seven increasingly regulated world. Annu. Rev. Entomol. Asclepias species and Calotropis procera. 51:45-66. Phytochemistry, 21:2343-2348. Konno, K., Ono, H., Nakamura, M., Tateishi, K., Singh, R. P. 1996 Monograph on promising pest control Hirayama, C., Tamura, Y., Hattori, M., Koyama, A., plant species of Asia and the pacific. Food and & Kohno, K. 2006. Mulberry latex rich in Agricultural Organization of the United Nations antidiabetic sugar-mimic alkaloids forces dieting Regional Office for Asia and the Pacific (RAP), on caterpillars. Proc. Natl. Acad. Sci. U S A., Bangkok, Thailand, pp. 37-38. 103:1337-1341. WHO,1981. Instructions for determining the Larhsini, M., Bousad, M., Lazrek, H.B., Jana, M. & susceptibility or resistance of mosquito larvae to Amarouch, H. 1997. Evaluation of antifungal and insecticides. Document WHO/VBC/81, 807, World molluscicidal properties of extracts of Calotropis Health Organization, Geneva. procera. Fitoterapia 68:371-373. Zalucki, M.P. & Brower, L.P. 1992. Survival of first Markouk, M., Bekkouche, K., Larhsini, M., Bousaid, M., instar larvae of Danaus plexippus (Lepidoptera: Lazrek, H. B. & Jana, M. 2000. Evaluation of some Danainae) in relation to cardiac glycoside and Moroccan medicinal plant extracts for larvicidal latex content of Asclepias humistrata activity. J. Ethnopharmacol. 73:293- 297. (Asclepiadaceae). Chemoecol. 3:81-93. Mwine, J., Van Damme, P. & Jumba, F. 2010. Evaluation of larvicidal properties of the latex of Euphorbia tirucalli L. (Euphorbiaceae) against larvae of Anopheles mosquitoes. J. Med. Plants Res. 4:1954-1959. Nishida, R. 2002. Sequestration of defensive substances from plants by Lepidoptera. Ann. Rev. Entomol. 47:57-92. Ramos, M.V., Bandeira, G.P., de Freitas, C.D.T., Nogueira, N.A.P., Alencar, N.M.N., de Sousa, P.A.S. & Carvalho, A.F.U. 2006. Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn). Mem. Instit. Oswaldo Cruz. 101:503-510.
Manuscript received on 23.10.2010, Accepted on 18.04.2011