The effects of an organophosphate insecticide on two enzyme loci in the shrimp Caradina sp.

BiochemicalSystematicsand Ecology,Vol. 20, No. 2, pp. 89-97, 1992. Printed in Great Britain.

0305-1979/92 $5.00+0.00 © 1992 Pergamon Press plc.

The Effects of an Organophosphate Insecticide on Two Enzyme Loci in the Shrimp Caradina sp. J. M. HUGHES, M. W. GRIFFITHS* and D. A. HARRISON Division of Australian Environmental Studies, Griffith University, Nathan, Queensland 4111, Australia

Key Word Index--Caradina sp.; tolerance-pollution monitoring.

freshwater shrimps;

pesticide-allozyme electrophoresis; differential

Abstract--This paper examines the effect of acute exposure to the organophosphate pesticide, chlorpyrifos, on genetic structure of freshwater shrimps. Genotype frequencies at two enzyme loci, Pgi and Pgm, were examined. There were significant differences in genotype frequencies between tolerant and susceptible animals in some experiments and not in others. This suggests that the differential tolerance occurs only at some concentrations or in some populations due to different linkage relationships between populations. The potential for using this method for monitoring pollution impacts is discussed.

Introduction Recent studies have indicated that environmental pollution is likely to reduce genetic variability within populations (Newman etaL, 1989; Battaglia etaL, 1980; Fevolden and Garner, 1987). Studies with particular pollutants have shown differential mortality between allozyme genotypes in freshwater fish (Chagnon and Guttman, 1989) and marine invertebrates (Lavie and Nevo, 1982; Lavie and Nevo, 1986; Nevo et al., 1981; Nevo et aL, 1983). Most of these studies have used heavy metals as the pollutants, although some earlier work investigated effects of certain pesticides on enzyme genotypes of insects (Beranek, 1974; Pasteur and Sinegere, 1975). These observations indicate that genetic structure of populations may be useful as a tool in monitoring environmental pollution. If particular genotypes are found to be differentially sensitive to particular pollutants, then reduced frequencies of those genotypes could be used as indicators of higher than usual levels of that pollutant, either currently or in the past. Obviously, other factors which affect genetic structure of populations, such as founder effects, genetic drift, gene flow and other environmental variables would need to be taken into account. The greatest potential for using such a technique is in monitoring effects of pollution within populations and their recovery through time. The properties that are important for a species to be considered as a genetic monitor are: (i) a wide distribution, so that results from one or a few sites can be extrapolated to a wider area; (ii) high density, so that a sufficiently large sample can be taken to calculate genotype frequencies; (iii) relatively low mobility, so that effects of pollution are not swamped by migration from other populations; (iv) sufficient variability at some enzyme loci, so that any selective effects of pollution can be recognized. Two freshwater species were chosen for investigation, both of which satisify the criteria outlined above. The mosquitofish (Gambusia affinis) is very resistant to a number of pollutants while the shrimp Caradina sp. is much more sensitive, particularly to pesticides (unpublished data). These two species were chosen so that both extremes of the tolerance range could be considered. The results for Gambusia are reported elsewhere (Hughes et aL, 1991). *Present address: Department of Entomology, University of Queensland, St. Lucia, 4067, Queensland, Australia.

(Received 8 October 1991) 89

J. M. HUGHES ETAL.

In this paper we report the results of an investigation of the effects of the organophosphate pesticide, chlorpyrifos, on two enzyme loci, Pgi and Pgm, in the shrimp Caradinasp. (family Atyidae). Materials and Methods The pesticide examined in the study, chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate), is an organophosphate which is replacing the more toxic organochlorines in the control of sugarcane beetles, termites and mosquitoes in Australia, and is being used in other parts of the world for mosquito and termite control (Schaefer and Dupras, 1970). It is known to be highly toxic to aquatic organisms (Connell and Miller, 1984). Experimental techniques. Shrimps were collected from two sites at Griffith University (27° 32' S, 153° 4' E), AES pond and GU dam, and one site on the other side of Brisbane, Moggill Creek (27° 31' S, 152° 55' E). They were collected using a dip net and were maintained in aquaria containing aged tap water for 24-28 h before being used in experiments. They were not fed either before or during experiments. Preliminary trials indicated that the 96-h LCs0was between 0.001 and 0.01 ppm chlorpyrifos. Seven separate experiments were run, with shrimps from different sites and with a range of chlorpyrifos concentrations. The collection site and the concentration of chlorpyrifos used in each experiment are shown in Table 1. We used a range of concentrations because it was possible that any differential survival among genotypes may differ between concentrations. Each of the first six experiments examined animals from only one site at a time, The first five experiments examined animals from GU Dam. Experiment 6 used animals from AES Pond. The final experiment attempted to compare responses from three sites by testing animals from all three simultaneously. Toxicity tests were performed using methods very similar to those outlined by Nevo et al. (1983). For each population, 30-50 shrimps were randomly assigned to a control tank. These shrimps were maintained in pure water for the duration of the experiment. If more than 10% mortality occurred in these controls the experiment was abandoned. The remaining shrimps were subjected to the chlorpyrifos, which was added to the tank water. Sample sizes differed among experiments, according to availability of shrimps. The tanks were checked regularly and dead animals were removed immediately and frozen at 80°C. For each of the first six experiments, this procedure was continued until approximately 50% of individuals had died. At this time the experiment was terminated and the survivors were also frozen. The first set of shrimps, i.e. those dying during the experiment, was labelled "susceptible" and the second set, i.e. those alive at the termination of the experiment, "tolerant". The final experiment was continued until all shrimps had died and they were divided into five time intervals, those dead after 2, 4, 8, 12 and 17 h, respectively. In the first five experiments, individuals were divided into two categories: gravid and non-gravid. In the sixth and seventh experiments they were measured before being analysed electrophoretically and divided into two size classes. Electrophoretic analysis. Each shrimp was homogenized in 0.4 ml of buffer (0.02 M "Iris, 0.001 M EDTA, 0.01 M NH4CI, 0.1 M glucose and 0.02% sodium azide), using an Ultra Turrax homogenizer, with 10G shaft. Samples were spun at 4°C at 10,000 rpm for 20 min in a Sorvall RC5B refrigerated centrifuge. The supernatant was decanted into vials and used immediately. Electrophoresis was performed on cellulose acetate plates (Titan 111, Helena Laboratories), using 75 mM Tris-citrate, pH 7.0, as electrode buffer. Two enzymes were examined, glucose phosphate isomerase (PGI, EC 5.3.1.9) and phosphoglucomutase (PGM, EC 2.7.5.1 ). Only one PGI locus was identified, for which there were four alleles. Two loci were identified for PGM but only one, Pgm-1, gave consistently readable results. There were three alleles at this locus. All animals were analysed for both enzymes but the numbers for the two enzymes are not equal in all cases because the patterns for particular individuals were not readable. For example, in Experiment 2, two whole plates stained for Pgidid not give readable results. Statisticalanalysis. Loglinear analysis (Feinberg, 1977) was used to analyse the data from each toxicity test. Separate three-way Ioglinear analyses were performed for each of the first six experiments. In Experiments 1-5, the three factors examined were (i) tolerance (i.e. susceptible or tolerant as defined above); (ii) reproductive condition (gravid or non-gravid); and (iii) a genetic factor (genotype, allele or heterozygote). In Experiments 6 and 7, size, rather than reproductive condition, was included as a factor. Some pooling was necessary to achieve sufficiently large numbers in individual cells. For Pgm, the 23,24,33 and 34 genotypes were pooled in the genotype analyses and the 2 and 3 alleles were pooled in the allele analyses. For Pgithe genotype 33 was compared against all others and the allele 3 compared against all others. For Experiment 7, four-way Ioglinear analyses were used, including site as an additional factor. As the main interaction of interest was time to death versus the genetic factor, generally only those higher order interactions involving these two factors were examined further. Whenever there was a significant three-way interaction involving time to death and a genetic factor, separate two-way interactions (within each level of the third factor) were examined for genotype, allele or heterozygote versus time to death.

Results

Experiments 1-6 The results for the first six experiments are presented in Tables 2 and 3. No samples

GU Dam April, 1988

GU Dam May, 1988

AES Pond Oct., 1988

4

5

6

0.003 32 h

0.005 36 h

0.01 7 h

0.01 6 h

Rep. cond. = reproductive condition.

Pgm genotype x rep. cond Pgm genotype

P=0.07 P<0.01

P - 0.07 P = 0.08

P-0.13 P<0.05 P<0.07 P<0.025

rep. cond × Pgm geootype cond. X Pgm allele

Tolerance x Pgm genotype × Within non-gravid: tolerance 44s more tolerant Tolerance x Pgm allele x rep. Within non-gravid: tolerance

Pgm heterozygote

Heterozygotes (34s) more susceptible Tolerance x Pgi genotype × size Within 33s and 34s: tolerance x size Large animals more tolerant

Tolerance x

None

None

P<0.05 P<0.05

P=0.08

P = 0.09

Tolerance x Pgi genotype 33s more tolerant

4s more tolerant, 3s more susceptible

P = 0.07

Tolerance × Pgi genotype 33s more susceptible

Homozygotes more tolerant

Within gravid: Tolerance × 4s more tolerant, 3s more susceptible Tolerance x Pgm heterozygote x rep. cond. Within gravid: tolerance × Pgm heterozygote

rep. cond.

P = 0.05 P<0.05

Level of significance

Pgm allele x Pgm allele

44s more tolerant, 34s more susceptible tolerance x

Within gravid: tolerance x

Tolerance x

Interaction

Only interactions which include a genetic factor and have probability levels less than 0.15 are shown.

GU Dam Ma~h, 1988

3

0.001 36 h

0.01 42 h

GU Dam March, 1988

GU Dam Ma~h, 1988

concentration (ppm) and length of exp.

2

Experiment

Collection site and date

Chlorpyrifos

TABLE 1. RESULTS OF LOGLINEAR ANALYSES ON TOXICITY TESTS

92

J. M, HUGHES ETAI

TABLE 2. PROPORTIONSOF SUSCEPTIBLEAND TOLERANT GENOTYPESAT THE Pgm LOCUS Exp. no.

Rep. cond.

Tol.

23

24

33

34

44

1

Gravid Gravid Non Non Gravid Gravid Non Non Gravid Gravid Non Non Gravid Gravid Non Non Gravid Gravid Non Non Small Small Large Large

Susc. Tol. Susc. Tol. Susc. Tol. Susc. Tol. Susc. Tol. Susc. Tol. Susc, Tol. Susc. ToI. Susc. Tol. Susc. ToI. Susc. Tol. Susc. Tol.

0.00 0,00 0.00 0.05

0.09 0.00 0.08 0.00

0.01 0.06

0.00 0.15 0.06 0.05

0.04 0.07 0.04 0.10 0.09 0.08 0.09 0.06 0.09 0.11 0.12 0,06 0.11 0.07 0.11 0.10 0.14 0.16 0.21 0.24 0.13 0.15 0.07 0.09

0.07 0.28 0.52 0,55 0.55 0.37 0.59 0.61 0,42 0.47 0.57 0.43 0.22 0.31 0.42 0.51 0.50 0.47 0.46 0.49 0.54 0.37 0.46 0.29

0.30~ 0,65 0.36 0.30 0.36 0.44 0.32 0.33 0.49 0,42 0,31 * 0.51 0.66 0.46 0.40 0.28 0.36 0,37 0.30 0.27 0.33 0.46 0.46 0.62

2

3

4

5

6

0.03 0,00 0.00 0.02 0.01 0.00

N 23 28 25 20 22 27 22 18 33 45 117 51 9 13 120 85 28 62 56 37 24 62 61 21

* = Significant (P<0.05) difference between Susceptible and tolerant in the frequency of that genotype, for the particulal reproductive condition (Test of Proportions, Freund, 1979); Rep. cond. = reproductive conditions; Tol.=tolerant; Susc. = susceptible.

TABLE 3. PROPORTIONS OF SUSCEPTIBLEAND TOLERANT GENOTYPESAT THE PgiLOCUS Exp. no.

Rep. cond.

Toi.

33

34

35

36

44

1

Gravid Gravid Non Non Gravid Gravid Non Non Gravid Gravid Non Non Gravid Gravid Non Non Gravid Gravid Non Non Small Small Large Large

Susc. Tol. Susc. Tol. Susc. Tol. Susc. Tol. Susc. Tol. Susc. Tol, Susc. Tol. Susc, ToI. Susc, Tol. Susc. Tol. Susc. Tol. Susc. Tol.

0.57 0,50 0.77* 0.47 0.40 0.60 0.47 0.71 0.50 0.53 0.45 0.39 0.90 0.92 0.75 0.75 0,54 0.47 0.51 0.54 0.25 0.43 0.57 0.48

0.17 0.12 0.04 0.21 0.07

0.09 0.23 0.15 0.16 0.13 0.23

0.04

0.04 0.04

2

3

4

5

6

0.37 0.07 0.19 0.27 0.31 0.35 0.10

0.14 0.22 0.16 0.17 0.16

0.15 0.18 0,18 0,15 0.24 0.27 0.25 0.22 0.26 0.10

0.09 0.04 0.04 0.16 0.05 0.08 0.25 0.10 0.03 0,14

0.05 0.07 0.10

45

0.05 0.20

46 0.09 0.11 0.04 0.05 0.13 0.03

Other

0.03

0.16 0.07 0.09 0.04 0.07 0.10

0.21 0.11 0.13 0.08 0.17 0.11 0.07 0.24

0.08 0.01 0.02 0.04 0.02 0.04 0.03 0.08 0.06 0.05

0.02

0.05 0.03

0.03

0.03 0.02

0.03 0.04 0.04

N 23 26 26 19 15 22 19 14 32 45 115 51 9 13 120 85 28 61 55 37 24 63 62 21

* = Significant (P<0.05, Test of Proportions, Freund, 1979) difference in genotype frequency between susceptible and tolerant groups, for that particular reproductive condition/size. Abbreviations as for Table 2.

EFFECTS OF INSECTICIDEON CARADINA

93

deviated from levels of heterozygosity expected under Hardy-Weinberg Equilibrium. A summary of significant interactions, determined from the Ioglinear analyses, is presented in Table 1. In only two of the six experiments was there a significant interaction between genotype and tolerance. At the Pgm locus, significant interactions between at least one genetic factor and tolerance were observed in Experiments 1 and 3. In Experiment 1, this interaction was observed only among gravid animals and in Experiment 3 it was only seen in non-gravid animals. In Experiment 6, the effect approached significance for all animals (P = 0.08). In each case there was a tendency for a higher frequency of the 44 genotype in the tolerant group and a higher frequency of 34s in the susceptible group (Table 2). As well in Experiments 1 and 3, there was a higher frequency of the 4 allele in the tolerant group and a higher frequency of the 3 allele in the susceptible group. The only other significant (P<0.05) interaction including a genetic factor was between tolerance, size and Pgigenotype in Experiment 6. This was caused by the fact that size affected tolerance within the 34 and 44 genotypes for Pgi (P<0.05), but not within the others, In Experiments 1 and 2, interactions between genotype and tolerance were significant at the 90% level (P<0.10), with 33s more susceptible than other genotypes in Experiment 1 and less susceptible in Experiment 2 (Tables 1 and 3).

Experiment7 The overall genotype frequencies at the three sites are shown in Tables 4 and 5. For both Pgi and Pgm, the Moggill Creek population was the least variable. For Pgl; all individuals, except one, were either 33s, 36s or 66s, whereas at the other two sites, although 33s were still the most frequent, 34s, 35s and 36s occurred in similar frequencies. For Pgm, the difference was more marked. At Moggil Creek, 98% of individuals were 33s whereas at the other two sites, 34s and 44s were more common than 33s. The three populations also differed in tolerance to chlorpyrifos (Table 6). The GU Dam and AES Pond populations appeared more sensitive, with 31% and 57%,

TABLE 4. PgiGENOTYPEFREQUENCIESAT THE THREE SITES IN EXPERIMENT 7

GU Dam AES Pond Moggill Creek

33

34

35

36

44,45,66

55,56,66

Total

0.62 0.62 0.69

0.10 0.14 0.00

0.11 0.07 0.06

0.11 0.10 0.27

0.02 0.05 0.00

0.03 0.03 0.04

271 172 169

TABLE 5. Pgm GENOTYPE FREQUENCIESAT THE THREE SITES IN EXPERIMENT 7

GU Darn AES Pond Moggill Creek

22

23

24

33

34

44

Total

0.00 0.00 0.00

0.00 0.01 0.00

0.04 0.02 0.00

0.16 0.11 0.98

0.40 0,52 0,02

0.43 0.35 0.00

257 172 159

TABLE 6. THE PROPORTIONOF EACH POPULATION DYING IN EACH TIME INTERVAL IN EXPERIMENT7

GU Dam AES Pond Moggill Creek

2h

4h

8h

12 h

17 h

Total

0.12 0.27 0.00

0.19 0.30 0.07

0.66 0.40 0.93

0.02 0.02 0.00

0.00 0,01 0.00

294 198 180

J.M. HUGHES ETAL

94

respectively, dying after 4 h. Only 6% of the Moggill population had died after 4 h. After 8 h almost all animals had died. Thus, the variability within populations in tolerance was greater in the GU Dam and AES Pond populations than in the Moggill Creek population. The fact that 98% of the Moggill Creek animals died in the same time period made it impossible to differentiate between "tolerant" and "susceptible" individuals. This population was excluded from further analysis. Because of small numbers in some categories, the data were pooled for further analysis. The times of death were 2 h, 4 h and greater than 4 h. There were two size classes, small and large. For Pgm,there was no significant four-way interaction, and no significant three-way interaction involving a genetic factor. The only interaction involving a genetic factor was a two-way interaction between site and genotype, with the AES pond population having a higher frequency of 34s than the GU Dam population (P<0.05). There were also no significant interactions involving Pgm allele or heterozygote (P>0.10). For Pg~there was no sigificant four-way interaction (Table 7). There was a significant three-way interaction between site, time and genotype. This interaction was further investigated by examining the two-way interactions between time and genotype for each site. Both were significant, but the effects observed were different. In the GU Dam population, for both size classes there was a higher proportion of 33s dying at time 2 than either at time 1 or time 3 (Fig. la, b). In the AES Pond population the highest proportion of 33s died at time 3 (Fig. lc, d). Similar results were observed for both allele and heterozygote analyses. The 3 allele was in highest frequency in the sample dying at time 2 for the GU Dam population and at time 3 for the AES Pond population. The proportion of heterozygotes was lowest in animals dying at time 2 in the GU Dam population and there was no significant interaction between time and heterozygote for the AES Pond population (Table 7).

Discussion In three of the first six experiments there was an indication of a relationship between Pgm and tolerance and in each case the same relationship was indicated. The trend

TABLE 7. RESULTSOF FOUR-WAY LOGLINEARANALYSESEXAMINING THE RELATIONSHIPBETWEEN SITE, SIZE, TOLERANCE AND ENZYME GENOTYPE,ALLELE AND HETEROZYGOTEAT THE PgiLOCUS

df

X2

(a) Genotype: Site× sizeXtirne×genotype

2

1.72

Sites×time×genotype GU Dam: timeXgenotype AES Pond: time×genotype

2 2 2

30.41 16,25 13,25

(b) Allele SiteX size×time ×allele

2

1.44

Site×time×allele GU Dam: time×allele AES Pond: timeXallele

2 2 2

26.98 16.47 12.59

(c) Heterozygote Site×size×time × heterozygote

2

2.54

Site×timeX heterozygote GU Dam: time×heterozygote AES Pond: timeXheterozygote

2 2 2

15.29 12.36 3.54

Level of significance

n.s. P<~O.0Ol P<0.001 P<~O.Ol n.s. PP>0.05

Because of low numbers in some cells, genotypes were pooled together. Only two genotypes were considered: 33s and "the rest". Similarly only two alleles were considered: 3s and the rest. The values for the four-way interactions are given, but then only those significant interactions involving the genotype, allele or heterozygote factor.

EFFECTS OF INSECTICIDE ON CARADINA

95

(a)

(c) I.O 0.8

b" c

~ h

~'

0.4

0,4

0.2

0.0

°+I/hL 0.6

0.6

lira 33

RI

mlI 34

35

0.2

im other

00

Genotype

(b)

(d) 1.0

53

34 35 Genotype

other

1.0 --

o.+ ~r

o" 0.4

~o4

o.o

Ihllr~l 33

34

I 35

Genotype

other

o,o

I 33

I II73~ I 34

35

other

Genotype

FIG. 1. GENOTYPE FREQUENCIES AT THE PgiLOCUS IN SHRIMPS FROM GU DAM AND AES POND THAT DIED AFTER TWO (BLACK), FOUR (WHITE) AND MORE THAN FOUR (HATCHED) HOURS, RESPECTIVELY. (a) GU Dam, small animals, (b) GU Dam, large animals, (C) AES Pond, small animals, (d) AES Pond, large animals.

was for the 44s and individuals carrying the 4 allele to be more tolerant than other genotypes. Although no significant interaction between Pgm and time to death in Experiment 7 was picked up in the Ioglinear analysis, some differences were observed for the GU Dam population. Within large animals, 44s made up 19% of the time 1 sample, 45% of the time 2 sample and 44% of the time 3 sample, indicating again that they tended to be more tolerant to the chlorpyrifos. Possibly, the different levels of response could result from different concentrations and the fact that animals were collected at different times of year. Only one significant (P
96

J.M. HUGHES ETAL.

at the Pgi locus would be likely to coincide with low variation in tolerance. An alternative explanation is that the lower variation in Pgl; also seen in Pgm, indicates overall low genetic variability in the population. This may also result in lower variability among individuals in their tolerance to chlorpyrifos. The fact that at both sites, genotype frequencies in animals dying at time 2 are not between those for times 1 and 3, is difficult to explain. Possibly some other mechanism of tolerance interacts in some way with Pg~ but only has an effect either immediately or after longer exposure. Obviously further work is needed to understand this effect. It may also explain the inconsistencies among Experiments 1-6, but because we only have two tolerance classes this cannot be assessed. The results of this study indicate that genetic variation at the Pgiand Pgm loci could not be used reliably to monitor pollution effects on a spatial scale. The effect on the Pgl locus was affected by the location from which the sample population was taken, implying a linked gene, rather than Pgigenotype itself affecting susceptibility. It may be possible to use Pgito monitor pollution effects on a temporal scale. If, in a particular population, the relationship between Pg/and susceptibility to chlorpyrifos is known, then changes in genetic structure following a pollution event, such as the spraying of nearby sugar cane, could be used to indicate the effects of the pollution. Due to the high susceptibility of shrimps to organophosphate pollution, significant changes in the shrimp Pgi genotype frequencies are likely to precede changes in other more tolerant species. Other workers (e.g. Nevo eta/., 1981, 1983; Lavie and Nevo, 1982, 1986; Diamond et a/., 1989; Benton and Guttman, 1990), working mostly with heavy metal pollution, have reported much more consistent results than those reported here, although it should be pointed out that not all these experiments report results from more than a single population. The possibilty of the results reported in these studies being due to linkage, rather than a direct effect on the enzyme being analysed, cannot be discounted. It seems likely that the lack of consistent relationships between tolerance and enzyme genotype is due to the lack of a direct effect of the pollutant on the enzymes being studied. The only studies in the literature that report a significant relationship between tolerance to pesticides and allozyme frequencies are those which investigated esterase loci (e.g. Tsakas and Krimbas, 1970; Beranek, 1974; Pasteur and Sinegere, 1975). Esterases play a role in the metabolic breakdown of pesticides within the organism (Baker, 1982; Beranek, 1974) and thus would be expected to be affected directly by the pollutant. Although esterase is polymorphic in Caradina sp., it is not possible to examine differences between tolerant and susceptible esterase genotypes in the same way as reported in this paper, because chlorpyrifos in even sublethal concentrations, inhibits enzyme activity so that genotypes cannot be identified. This effect is the subject of further work and will be reported in a future paper. Acknowledgements--This project was funded as part of the core programme of the Centre for Catchment and Instream Research, which is funded by the Australian Water Resources Advisory Council.

References Baker, J. (1982) Selective effects of insecticides on within-species variation: the lessons to be learnt when considering the environmental effects of pollution. Agric. Env. 7, 187-198. Battaglia, B., Bisol, P. M. and Rodino, E. (1980) Experimental studies on some genetic effects of marine pollution. Heligolander Meeresuntersuchungen 33, 587-595. Benton, M. J. and Guttman, S. I. (1990) Relationship of allozyme genotype to survivorship of mayflies (Stenomena femoratum) exposed to copper. J. N. Am. Benthol. Soc. 9, 271-276. Beranek, A. P. (1974) Esterase variation and organophosphate resistance in populations of Aphis fabae and Myzus persicae. Entomol. Experiment. Applic. 17, 129--142. Chagnon, N. L. and Guttman, S. I. (1989) Biochemical analysis of allozyme copper and cadmium tolerance in fish using starch gel electrophoresis. Env. Toxicol. Chem. 8, 1141-1147. Connell, D. W. and Miller, G. J. (1984) Chemistry and Ecotoxico/ogy of Pollution. John Wiley, Brisbane.

EFFECTSOF INSECTICIDEON CARADINA

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Diamond, S. A., Newman, M. C., Mulvey, M., Dixon, P. M. and Martinson, D. (1989) Allozyme genotype and time to death of mosquitofish, Gambusia affinis (Baird and Girard) during acute exposure to inorganic mercury. Env Ecotoxicol. Chem. 8, 613-622. Feinberg, S. E. (1977) The Analysis of Cross Classified Categorical Data. M.I.T. Press, London. Fevolden, S. E. and Garner, S. P. (1987) Environmental stress and allozyme variation in Littorina littorea (Prosobranchia). Mar. Ecol. Prog. Ser. 39, 129-136. Freund, J. E. (1979) Modern Elementary Statistics. Prentice Hall, Sydney. Gillespie, R. B. and Guttman, S. I. (1989) Effects of contaminants on the frequencies of allozymes in populations of the central stoneroller. Env. Toxicol. Chem. 8, 613-622. Hughes, J. M., Harrison, D. A. and Arthur, J. M. (1991) Genetic variation at the Pgi locus in the mosquito fish Gambusia affinis (poccilidae) and a possible effect on susceptibility to an insecticide. Biol. J. Linn. Soc. 44, 153-167. Lavie, B. and Nevo, E. (1982) Heavy metal selection of phosphoglucose isomerase allozymes in marine gastropods. Ma~ Biol. 71, 17-22. Lavie, B. and Nevo, E. (1986) Selection against rare alleles in mercury pollution: multilocus structure across five gastropod species. In Environmental Quality & Ecosystem Stability (Dubinsky, Z. and Steinberg, V., eds). Ramat-Gan, Bar-lnhlan Press, Israel. Nevo, E., Perl, T., Belles, A. and Wool, D. (1981) Mercury selection of allozyme genotypes in shrimps. Experientia 37, 1152-1154. Nevo, E., Perl, T. and Ben-Shlomo, R. (1983) Selection of allelic isozyme polymorphisms in marine organisms: pattern, theory and application. Isozymes, Curr. Top. Biol. Med. Res. 10, 69-92. Newman, M. C., Diamond, S. A., Mulvey, M. and Dixon, P. (1989) AIIozyme genotype and time to death of mosquitofish, Gambusia affinis (Baird and Girard), during acute toxicant exposure: a comparison of arsenate and inorganic mercury. Aq. Toxicol. 15, 141-156. Pasteur, N. and Sinegere, G. (1975) Esterase polymorphism and sensitivity to Dursban organophosphorous insecticide in Culex pipens pipens populations. Biochem. Gen. 13, 789-803. Schaefer, C. H. and Dupras, E. F. (1970) Factors affecting the stability of Dursban in polluted waters. J. Econ. Ent. 63, 701-705. Tsakas, S. and Krimbas, C. B. (1970) The genetics of Dacus oleae: IV. Relation between adult esterase genotype and survival to organophosphate insecticides. Evolution 24, 807-815.