Artificial incubation of the eggs of the crayfish Austropotamobius pallipes (Lereboullet)

Aquaculture, 25 (1981) 129-140 Elsevier Scientific Publishing Company,

ARTIFICIAL

INCUBATION

A USTROPOTAMOBIUS

129 Amsterdam

- Printed

in The Netherlands

OF THE EGGS OF THE CRAYFISH (LEREBOULLET)

PALLIPES

C.P. RHODES Department

of Zoology, The University, Nottingham,

Present address: (Great Britain) (Accepted

Department

of Zoology,

University

NG7 2RD (Great Britain) College Swansea,

Swansea

SA2 SPP

10 March 1981)

ABSTRACT Rhodes, C.P., 1981. Artificial incubation of the eggs of the crayfish pallipes (Lereboullet). Aquaculture, 25: 129-140.

Austropotamobius

In Great Britain, incubation time for eggs of the native crayfish, Austropotamobius pallipes, is approximately 9 months, with hatching occurring in May and June in Midlands waters. Artificial incubation at higher than ambient temperature would possibly reduce this time and make juveniles available for stocking in early spring. To test this hypothesis, fertile eggs were removed from the pleopods of ovigerous females maintained at ambient temperatures at different times (30-day intervals from late November through late April) and incubated in flowing water baths at 7 +_l”C, 13 + 1°C and 18 f 1°C. Only eggs stripped from females from late January-April survived to hatching in the artificial incubation system, and none survived at 7 + 1°C. However, incubation at 13 + 1°C and 18 ?-1°C produced large numbers of juveniles as early as March and April. Overall, 13 i 1°C was the best incubation temperature, considering both survival to hatching (average of 72% from February through April) and early development of the hatchlings. Daily dosing of the eggs with malachite green (10-20 ppm) eliminated fungal infections from the artificial incubation systems. INTRODUCTION Interest and

Fuke,

in crayfish 1977;

farming

Behrendt,

in Great 1979;

Rhodes

Britain and

is currently Holdich,

very 1979).

high

(Richards

Indeed,

this

may have been influenced by reports of high European and Scandinavian market prices (Karlsson, 1977; Fuke, 1978) and the belief that extra income may be extracted from crayfish stocked water bodies with little or no management, effort, (Karlsson, 1977). Although the possibility and consequences of commercial crayfish exploitation in Great Britain still require much empirical and quantitative assessment, recent reports indicating that our native British crayfish resources have been eradicated by the crayfish plague (Anonymous, 1976; Richards and Fuke, 1977; Karlsson, 1978) are far from accurate. Indeed, large and healthy populations of Austropotamobiuspallipes are known to occur in many parts of the British Isles (Holdich et al., 1978; Ingle, 1978; 0044-8486/81/0000-0000/$02.50

o 1981 Elsevier

Scientific

Publishing

Company

130

Jay and Holdich, 1981), and there is no evidence to support the contention that the plague disease is present within British waters (Bowler, 1979). The above misconception, in addition to a general paucity of information concerning the biology of Austropotamobius pallipes, has resulted in the import of alien crayfish species for release into British ecosystems. The current lack of import disease certification and its attendant ‘free traffic’ in both juvenile and possibly adult specimens is causing British crayfish biologists some concern (Holdich et al., 1978; Bowler, 1979; Goddard and Holdich, 1979; Rhodes, 1980). In view of the dangers associated with alien crayfish imports, it is thought expedient to bring to the attention of commercial concerns details of the unexploited native British crayfish resource and to examine its potential. Studies pertaining to reproductive biology and subsequent growth may be considered as primary among biological criteria necessary for an evaluation of a particular species for commercial concern (see Webber and Riordan, 1976). Although many valuable studies have been effective in elucidating details of the reproductive biology of Austropotamobius pallipes (see Rhodes (1980) for review), the possibility of artificially incubating eggs detached from the maternal female seems to have been entirely neglected. Any attempts to involve crayfish in a culture strategy or to raise large numbers of juveniles for stocking purposes clearly requires a degree of reproductive manipulation. The reduction of natural incubation time (approximately 9 months) effected by temperature manipulation is obviously highly desirable. The possibility of rearing large numbers of juvenile Austropotamobius pallipes within the British Isles appears not to have been previously examined. In the present series of experiments, both maintenance of egg bearing females at elevated temperatures and removal of pleopodal eggs in order to determine the earliest possible stripping date consistent with acceptable hatching results were performed. Details of artificial incubation methodology are given. METHODS

AND MATERIALS

Ovigerous females were noted to occur in the River Leen and the Nanpantan Reservoir study populations (see Rhodes and Holdich, 1979) from mid-October in both the 1977-1978 and the 1978-1979 seasons. All ovigerous females encountered at both sites during field collection trips throughout the entire season were sampled by hand-netting and returned to the laboratory. Crayfish in the 23.1-48.6 mm carapace length size range were included in the study (N = 123). Specimens were maintained in outdoor rearing tanks possessing an adequate supply of food and excess refugia until required for experimental purposes. Despite the observation that ovigerous females maintained at a constant 10 + 1°C successfully hatched their eggs following an incubation period comparable to that noted for field specimens, maintenance of berried females at the elevated temperatures of 13 + 1°C and 18 f 1°C invariably resulted in rapid

131

loss of all pleopodal eggs. Therefore, experiments employing this methodology were terminated following the 1977-1978 season, and experiments involving the removal of pleopodal eggs and subsequent artificial incubation at elevated temperatures were initiated. Egg

incubation

apparatus

The incubation apparatus (see Fig. 1) consisted of three individual chambers maintained at 18 t l”C, 13 f 1°C and 7 f l”C!, respectively. Each chamber received approximately 75 ml of water per minute and aeration was provided. Crayfish eggs were borne upon submerged nylon mesh trays. Each incubation chamber received approximately 220 lux on a 16 : 8 light to dark regime. In order to improve spectral quality, a ‘grow-lux’ light source was included in the time switch arrangement. Physicochemical and water quality data are shown in Table I. Pleopodal eggs were removed from berried females at 30-day intervals from 30 November to 29 April in the 1977-1978 season and from 28 November to 27 April in the 1978-1979 season, Ovigerous females were maintained in outdoor rearing tanks for at least one month prior to stripping. The effects of previous environmental history on the pleopodal eggs used in these experiments could not be controlled or interpreted. At each 30-day interval, 300 eggs (100 for each experimental temperature) were removed from at least six females including specimens from each collection site. Blunt forceps were used in the removal of pleopodal eggs. Detached eggs were allowed to fall into tanks containing large volumes of water at the outdoor

Fig. 1. Diagrammatic representation tion of the eggs of Austropotamobius

of the apparatus employed in the artificial pallipes. ht = header tank.

incuba-

132 TABLE I Physicochemical

and water quality data pertinent to artificial incubation

Parameter

Value

Temperature (“C) PH Dissolved oxygen (ppm) Total hardness (Ca & Mg) (ppm) Conductivity (pmho) Ammonia nitrogen (ppm) Calcium ion (ppm)

18+1,13~1,7*1 1.25 f 0.29 8.0 + 0.4 175 f 15 3102 10 < 0.1 50.0 + 3.0

48 48 24 24 24 24 24

ambient temperature, and to attain slowly the desired experimental temperature over at least one day. Selection of eggs for particular thermal regimes was random and only apparently healthy and undamaged eggs were included in the study. No large immediate post-stripping mortalities were recorded. Treatment of detached eggs to prevent fungal infection was found to be of paramount importance. Initial experiments during the 1976-1977 season revealed that the use of commercial preparations of Phenoxetol and natural control by the grazing of Asellus aquaticus and Gammarus pulex (see Oseid, 1977) were completely ineffective. However, daily dosing of the incubation trays using malachite green (lo-20 ppm) was found to eliminate fungal infection completely from the trays of developing eggs (see also Vey, 1977). Dead eggs (light brown/orange or yellow appearance) were removed daily and the number of surviving individuals noted. Successful incubation was defined as survival to hatching, although survival to semi-independent juveniles following a single moult was also recorded. The dates of hatching were noted for each batch of eggs. Hatchlings readily attached to nylon netting as a substitute for the maternal ventral abdominal surface (see Holdich et al., 1978; Rhodes, 1980). The effects of incubation temperature and time of stripping on survival and hatching success were examined by analysis of variance (see Parker, 1973) Duplicate results were included in the two-way analysis. In order to facilitate computation, the General Statistical Programme (GENSTAT V) software package (Lawes, 1977) was utilised. Tables of means were prepared and the standard errors of the differences of the means calculated. The differences between the means of survival at the various time and temperature treatments were tested for significance using the multiple ‘studentised’ range test procedure (see Zar, 1974). The number of days to first and last hatching of the various batches was calculated, and the range (in days) over which batch hatching took place noted. The number of days X degrees accumulated during incubation is presented. Where possible, attempts have been made to compare the present data with those obtained for other species of crayfish examined in a similar manner.

133 RESULTS

Survival of artificially incubated eggs of the crayfish Austropotamobius is shown in Table II. Data concerning survival of individuals to hatching and stage 2 semi-independent juveniles are presented. It is clear that eggs failed to hatch when maintained at 7 5 1°C. Failure to survive and hatch at this temperature was recorded for each of the six stripping times. Eggs removed at the November and December stripping times failed to hatch when incubated at the elevated temperatures of 13 f 1°C and 18 + 1” C. Successful hatching was achieved for the January strip, but percentage survival was poor, particularly at 18* 1 ‘C. Marked improvement in survival to hatching was noted in batches incubated from late February (strip 4) onwards. Up to approximately 90% hatching success was achieved with increasing closeness of the stripping date to the natural pleopodal hatching time. Continued dosing of the incubators with malachite green was found to be unnecessary following hatching, although egg stages were found to become rapidly infected without this treatment. Survival from the hatchling stage to stage 2 was noted to improve markedly (approximately 10% mortality between stages) for eggs removed from the February strip onwards. At 13 rt 1°C moulting was observed to take place normally, resulting in healthy stage 2 individuals. However, at 18 + 1°C many crayfish failed to moult successfully and were typically unable to shed completely their hatchling exuvium (numbers shown in brackets, Table II). Subsequent hardening of the new stage 2 exoskeleton resulted in severe deformation of the walking legs and other appendages_ Deformed individuals invariably died following several days enforced inactivity.

pallipes

TABLE II Numbers of survivors to hatching and stage 2 juveniles for replicate batches of 100 eggs stripped at six intervals and maintained at three incubation temperatures. Numbers of animals which failed to moult normally are shown in brackets. Survival to hatching Date -____ ..-- ._.__~ 30 Nov 1977 28 Nov 1978 30 Dee 1977 28 Dee 1978 29 Jan 1978 27 Jan 1979 28 Feb 1978 26 Feb 1979 30Mar 1978 28 Mar 1979 29 Apr 1978 27 Apr 1979

Survival to stage 2

Strip

7t 1°C

13* 1°C

18* 1°C

711°C

13i1°C

1 1 2 2 3 3 4 4 5 5 6 6

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 36 21 63 53 76 66 86 87

0 0 0 0 6 0 80 62 77 71 91 80

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 20 7 62 49 68 59 66 72

0 0 0 0 0 0 17 11 27 12 11 18

l&l%

(59) (48) (48) (55) (71) (57)

-

134

Data obtained on survival to hatching were subjected to an analysis of variance procedure (see Table III). The effects of time (strip), incubation temperature, and time and temperature combined on survival are clearly demonstrated. Analysis also revealed a significant difference between replicate data compiled in the 1977-1978 and 1978-1979 seasons, thus indicating that previous investigations concerning the artificial incubation of crayfish eggs (see discussion), which invariably rely upon a single year’s data, may not be absolute. In order to account for this difference, computation of the means of survival to hatching for both replicates, considering both time and temperature were undertaken. This clearly showed that the difference between replicates is accounted for by a lower survival in the 1978-1979 season. As experimental procedures were not varied, it must be assumed that natural variation was responsible for this difference. Examination of the mean survival to hatching at 13 i 1°C and 18 i 1°C over the total incubation period showed no significant difference when tested using a multiple range procedure (see Table III), For combined temperatures, no significant difference in TABLE III Analysis of the effects of temperature and time of stripping on the survival of eggs to hatching To Hatching ANOVAR SURVIVAL

DF

SS

SS%

MS

F(VR)

P

1. 2. 3. 4.

1 2 5 10 17

156.25 12685.39 20160.81 10891.28 315.25

0.35 28.69 45.60 24.64 0.71

156.25 6342.69 4032.16 1089.13 18.54

8.426 342.033 217.436 58.732

< < < <

Between replicates Temperature Time Temperature X time Residual

0.01 0.01 0.01 0.01

Table of means and multiple ‘studentised’ range test: Temperature 1. 2. 3. 7+ 1°C 13* 1°C 18* 1°C 0.00 40.67 38.92 Comparison 2 vs 3

Difference 1.75

SE 1.758

4 0.995

P 2

Time

1. 0.00

3. 10.50

4. 43.00

5. 48.33

Comparison 6 vs 3 6 vs 4 6 vs 5 5 vs 3 5 vs 4 4 vs 3

Difference 46.83 14.33 9.00 37.83 5.33 32.50

2. 0.00

SE 2.486 2.486 2.486 2.486 2.486 2.486

q 18.837 5.764 3.620 15.217 2.144 13.073

qO.O1,16,p 4.131

P 4 3 2 3 2 2

Significance N/S

6. 57.33 qO.O1,16,p 5.192 4.786 4.131 4.786 4.131 4.131

Significance < 0.01 < 0.01 N/S < 0.01 N/S < 0.01

135

survival to hatching could be detected between stripping times 6 and 5, and 5 and 4. Differences between other stripping times were apparent (see Table III). Analysis of variance and associated multiple range testing for survival to stage 2 juveniles is shown in Table IV. The significant effects of temperature and time of stripping on survival are again clearly demonstrated. %I difference between replicates was recorded. A clear difference in survival to stage 2 was shown between the 13 ? 1°C and the 18 + 1°C incubation temperatures (Table IV). Poor survival at 18 f 1 “C!resulting from moulting abnormalities undoubtedly influenced this outcome and dictates an incubation ambient of 13 t 1°C preferable for the successful rearing of stage 2 juveniles. Hatching of pleopodal eggs was observed to take place at approximately the same time in both the outdoor rearing tanks and at the field collection sites. Dates of hatching were 21-27 May and 16-22 June in the 1977-1978 and the 1978-1979 seasons respectively. Artificial incubation technique is clearly capable of producing healthy juveniles as early as March, with hatching TABLE IV Analysis of the effects of temperature and time of stripping on the survival of eggs to stage 2 juveniles To Stage 2 ANOVAR SURVIVAL

DF

SS

SS%

1. 2. 3. 4.

1 2 5 10 17

51.36 7385.39 5665.81 5716.61 331.14

0.27 38.57 29.59 29.85 1.73

Between replicates Temperature Time Temperature x Time Residual

MS _______ 51.36 3692.69 1133.16 571.66 19.48

F(VR) _ ~._~~_ 2.637 189.575 58.174 29.348

P N/S < 0.01 < 0.01 < 0.01

Table of means and multiple ‘studentised’ range test: Temperature 1 2. 3. 7 f 1°C 13? 1°C 18* 1°C 8.00 0.00 33.58 Comparison 2 vs 3

Difference 25.58

Time

1. 0.00

Comparison 6 vs 3 6 vs 4 6 vs 5 5 vs 3 5 vs 4 4 vs 3

Difference 23.33 4.66 0.16 23.17 4.50 18.67

2. 0.00

SE 1.802 3. 4.50 SE 2.548 2.548 2.548 2.548 2.548 2,548

q 14.195

;

4. 23.17

5. 27.67

q. 9.156 1.829 0.628 9.093 1.766 7.327

: 3 2 3 2 2

qO.O1,16,p 4.131

Significance < 0.01

6. 27.83 qO.O1,16,p 5.192 4.786 4.131 4.786 4.131 4.131

Significance < 0.01 N/S N/S < 0.01 N/S < 0.01

136 TABLE

V

Data pertaining

to the hatching

times of crayfish

eggs maintained

Date

Strip

Temp. CC)

Date first hatch

to

29 Jan

1978

3

13 18

12 Mar 24 Mar

27 Jan

1979

3

13 18

19 Mar

28 Feb

1978

4

13 18

26 Feb

1979

4

30 Mar

1978

28 Mar

at elevated

incubation

temperatures

_.___-

~. ..Date to last hatch _~ 1 Apr 10 Apr

Days. from strip to last hatch

Hatching range (days) ____

Days X degrees accumulated in incubators ____-

21 18

62 71

806 1278

9 Apr -

22 -

72

936 -

18 Mar 29 Mar

9 Apr 3 Apr

23 6

40 34

520 612

13 18

12 Mar 19 Mar

6 Apr 5 Apr

25 18

42 41

546 738

5

13 18

27 Apr 23 Apr

19 May 12 May

23 20

50 43

650 774

1979

5

13 18

17 Apr 10 Apr

12 May 21 Apr

26 i2

45 26

585 468

29 Apr

1978

6

13 18

15 May 13 May

30 May 22 May

16 10

31 23

403 414

27 Apr

1979

6

13 18

9 May 5 May

21 May 16 May

13 11

24 19

312 342

___-

of large numbers by April (Table V). The number of days for entire batches of eggs to hatch (hatching range in days) was smaller at the higher incubation temperature (Table V). A decreasing number of days hatching range with increasing lateness of strip was also noted. Incubation time to hatching decreased with increasing lateness of strip and batches of eggs removed on the same day usually hatched in less time at 18 f 1°C than at 13 f 1°C. In terms of days X degrees accumulated in the incubators, which does not take into account days X degrees accumulated prior to stripping, eggs maintained at 18 + 1°C usually accumulated more of these ‘units’ to hatching than did eggs incubated at 13 f 1°C. The processes of crayfish embryonic development may not be directly proportional to environmental temperature. DISCUSSION

A number of authors have been successful in shortening the period of natural pleopodal incubation by maintaining ovigerous female crayfish at elevated temperatures. Employing this methodology, juvenile specimens of the freshwater crayfish Pacifastacus leniusculus have been hatched 4-5 months earlier than under natural conditions (see Cabantous, 1975; Goldman et al., 1975; Strempel, 1975; Westman, 1975). Emadi (1974) found that eggbearing females of this species lost all or most of their eggs during confinement in individual culture cells. In a few, however, the egg carrying period was reduced by several months following maintenance at a constant 15°C.

137

Maintenance of berried specimens of Austropotamobius pallipes at elevated temperatures resulted in complete loss of pleopodal eggs. It seems likely that female behaviour or cement attachment substance was in some way adversely affected by temperature elevation. Indeed, Brown (1979) has noted that the initial egg attachment procedure may be less successful in the native crayfish at higher temperatures. Loss of maternal eggs at elevated temperatures suggests that the only rational means of shortening natural incubation time may be by artificial incubation technique. Indeed, artificial incubation of the eggs of Astacus astacus has been successfully attempted by Cukerzis (1973) and Strempel (1973). A hatching success rate of over 80% has been reported by these authors. More recently, Mason (1977) artificially incubating the eggs of Pacifastacus leniusculus has reported a 90-98% success rate to hatching and a 77-88s success rate to stage 2 juveniles. Despite being a ‘cold water’ species, failure of the eggs of Austropotamobius pallipes to survive or hatch when incubated at 7 * 1°C perhaps indicates the requirement of a temperature rise prior to hatching. Abrahamson and Goldman (1970) have reported a similar failure of the eggs of Pacifastacus leniusculus to hatch when kept at an average of 6.8”C throughout the brooding period. In the present series of experiments, artificial incubation at elevated temperatures has been shown capable of producing crayfish juveniles as early as March, with more substantial numbers hatching in early April. Removal of pleopodal eggs before late February (strip 4) was clearly disadvantageous. From late February and early March onwards, 18 f 1°C may be considered a more suitable incubation temperature to hatching. However, in view of the subsequent moult-related mortality at this temperature, 13 ? 1°C appears a more profitable incubation temperature in terms of the total number of healthy stage 2 individuals produced. Experiments to determine whether quantities of juveniles hatched at 18 t 1°C could be induced to moult successfully by lowering the incubation ambient to 13? 1°C during the hatchling intermoult are yet to be performed. Failure to escape from the hatchling exuvium and attendant deformity was only observed at 18 f 1°C. Mortality of this nature appeared specific to the initial moult and was infrequently observed during later moults. Dietary inadequacies have often been associated with incomplete ecdysis (see Wickins, 1972 ; Bayer et al., 1978). However, hatchlings are lecithotrophic prior to their first moult (see Rhodes, 1980) and should possess an adequate yolk store. Thermal damage, possibly manifested in disruption of exoskeletal synthesis or excessively rapid metabolism of essential yolk component, is clearly implicated. Koo and Johnston (1978) have reported larval deformities in fish due to heat damage of the developing eggs. Disruption of embryological cellular mechanics may have been implicated in this outcome. Phycomycete fungi of the family Saprolegniales are known to infect the eggs of both marine and freshwater crustaceans (Atkins, 1954; Vey and Vago, 1973; Vey, 1977). Infection’during incubation was easily identified by the

138

presence of white myceiial threads and the orange colour of the infected eggs. Tendency for fungal infection was high, particularly at elevated temperatures. However, it was not clear whether egg deaths were caused initially by the fungus or whether egg death mediated by some alternative cause was followed by fungal infection. Once established, disease rapidly spread throughout the entire batch. Use of malachite green was completely successful in preventing such fungal disease spread (see also Mason, 1977; Rehulka, 1977; Vey, 1977). Control of fungal disease using Asellus and Gammarus (see Oseid, 1977) and dosing with a commercial preparation of Phenoxetol was found to be entirely ineffective. Many females in outdoor rearing tanks were observed to lose eggs over the natural incubation period. However, data on maternal reproductive efficiency were not recorded during this series of experiments. Comparison of rearing success in terms of numbers of viable young produced between natural and artificial incubation methods was therefore not possible. Artificial incubation has, however, the clear advantage of producing juveniles at a relatively early time. Implantation of such early specimens into natural ecosystems would allow them to take full advantage of the spring/summer growth period prior to the onset of winter. Naturally brooded individuals may well be at a disadvantage in this respect. It is suggested that the incubation procedure described may be modified; other environmental parameters such as dissolved oxygen concentration require full appraisal. Any increased production efficiency resulting from artificial incubation must be balanced against increased costs. Subsequent manipulation of environmental parameters to achieve good growth and survival of reared juveniles requires much investigation. Work pertaining to such a consideration is presently being prepared. ACKNOWLEDGEMENTS

Many thanks are due to Professor P.N.R. Usherwood for the provision of excellent laboratory facilities and to Dr D.M. Holdich for many useful discussions during the course of this investigation. The work was financed by a grant from the Science Research Council.

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Behrendt, A., 1979. Export push needed to open market for crayfish. Fish Farmer, 2: 44-47. Bowler, K., 1979. Plague that has ravaged Europe. Fish Farmer, 2: 34-35. Brown, D.J., 1979. A study of the population biology of the British freshwater crayfish Austropotamobius pallipes (Lereboullet). Ph.D. Thesis, University of Durham, U.K., l-262. Cabantous, M.A., 1975. Introduction and rearing of Pacifastacus at the research center of les Clouzioux 18450 Brinons/Sauldre, France. Freshwater Crayfish, 2: 49-63. Cukerzis, J., 1973. Biologische Grundlagen der Methode der Kunstlichen Aufzucht der Brut des Astacus astacus L. Freshwater Crayfish, 1: 187-201. Emadi, H., 1974. Culturing conditions and their effects on survival and growth of the crayfish Pacifastacus Zeniusculus trowbridgii (Stimpson). Ph.D. Thesis, Oregon State University, U.S.A. Fuke, P., 1978. Crayfish: A E5000 per acre investment? Country Landowner, Feb/March XXIX: p. 20. Goddard, J.S. and Holdich, D.M., 1979. Explore the native potential first. Fish Farmer, 2: p. 47. Goldman, C.R., Rundquist, J.C. and Flint, R.W., 1975. Ecological studies of the California crayfish, Pacifastacus Zeniusculus, with emphasis on their growth from recycling waste products. Freshwater Crayfish, 2: 481-487. Holdich, D.M., Jay, D. and Goddard, J.S., 1978. Crayfish in the British Isles. Aquaculture, 15: 91-97. Ingle, R.W., 1978. The white footed crayfish. Underwater World, May/June: 20--21. Jay, D. and Holdich, D.M., 1981. The distribution of the crayfish Austropotamobius pallipes (Lereboullet) in British waters. Freshwater Biol., 11: 121-129. Karlsson, S., 1977. The freshwater crayfish. Fish Farming Int., 4( 2): 8-12. Karlsson, S., 1978. Open mind about crayfish. Fish Farmer, 1: p. 44. Koo, T.S.Y. and Johnston, M.L., 1978. Larval deformity in striped bass Morone saxatilis (Walbaum), and blueback herring Alosa aestiualis (Mitchill), due to heat shock treatment of developing eggs. Environ. Pollut., 16: 137-149. Lawes, 1977. Genstat V. Lawes Agricultural Trust (Rothamsted Experimental Station) ICL 1900 Conversion by Oxford University Computing Service. Mason, J.C., 1977. Artificial incubation of crayfish eggs Pucifastucus Zeniuscufus_ (Dana). Freshwater Crayfish, 3: 119-132. Oseid, D.M., 1977. Control of fungus growth on fish eggs by Asellus militaris and Gammarus pseudolimnaeus. Trans. Am. Fish. Sot., 106: 192-195. Parker, R.E., 1973. Introductory Statistics for Biology. The Institute of Biology Studies in Biology, No. 43, Edward Arnold, London. Rehulka, J., 1977. Pouziti snasenlivost a toxicita dlouhodobe loupele v malachitove zeleni b pro podzimni kapri pludek. Zivocisna Vyroba, 22: 711-720. Rhodes, C.P., 1980. Studies on the growth and feeding biology of the crayfish AustropotamobiuspoEZipes (Lereboullet). Ph.D. Thesis, University of Nottingham, U.K., 546 pp. Rhodes, C.P. and Holdich, D.M., 1979. On size and sexual dimorphism in Austropotamobius pollipes (Lereboullet). A step in assessing the commercial exploitation potential of the native British freshwater crayfish. Aquaculture, 17: 345-358. Richards, K. and Fuke, P., 1977. Freshwater crayfish: the first centre in Britain. Fish Farming Int., 4( 2): 12-15. Strempel, K., 1973. Edelkrebserbrutung in Zuger-Glasern und Anfutterung der Krebsbrut. Freshwater Crayfish, 1: 233-237. Strempel, K., 1975. Kunstliche Erbrutung von Edelkrebsen in Zugerglasern und Vergleichende Beobachtungen im Verhalten und Abwachs von Edel- und Signalkrebsen. Freshwater Crayfish, 2: 393-403. Vey, A., 1977. Studies on the pathology of crayfish under rearing conditions. Freshwater Crayfish, 3: 311-319.

140 Vey, A. and Vago, C., 1973. Protozoan and fungal diseases of Austropotamobius pallipes (Lereboullet) in France. Freshwater Crayfish, 1: 165-179. Webber, H.H. and Riordan, P.F., 1976. Criteria for candidate species for aquaculture. Aquaculture, 7: 107-123. Westman, K., 1975. On crayfish research in Finland. Freshwater Crayfish, 2: 65-75. Wickins, J.F., 1972. The food value of brine shrimp, Artemia salina L., to larvae of the prawn, F’alaemon serratus Pennant. J. Exp. Mar. Biol. Ecol., 10: 151-170. Zar, J.H., 1974. Biostatistical Analysis. Prentice-Hall, Inc., Englewood Cliffs, NJ.