RADIATION DISINFESTATION STUDIES ON SUN-DRIED FISH

Mainuddin Ahmed, A.D. Bhuiya, AMS. Alam and S.M.S. Huda Irradiation and Pest Control Research Institute (IPCORI) Bangladesh Atomic Energy Commission Dacca-2, Bangladesh

The loss of a large quantity of dried fish in Bangladesh is due to infestation by earwigs, hide and copra beetles in storage godowns. The most destructive pest is the hide . Dennestes maculatus Degeer.

Earwigs of different stages of development were exposed to 10, 20 and 30 krad of gamma radiation for disinfestation. No live were seen 3 weeks after the treatment. Studies on dried mackerel infested with hide beetles showed that eggs, larvae and pupae could be checked at a dose of 20 krad. A dose of 30 krad killed all adults within 2 weeks.

In packaging studies, dried mackerel was packed in polythene pouches of different thicknesses. All irradiated treatments were free from damage while the controls were heavily damaged. However, all these treatments developed heavy fungdl growth except the heat-treated control. in other experiments, pouches of 50,75 and 100 y thickness were exposed to 50, 100,200 and 400 krad as in the previous exper- iment. In all the treatment, moistures content was reduced to 13 percent. Heavy insect damage was ob- served in the control but all other treatments were in excellent condition after 5 months' storage.

INTRODUCTION

Sundried, smoked, salted and dried fish are favourite foods in South East Asia. In Bangladesh freshwater fis'h is preferred; during peak periods, the surplus fish is sundried for offseason consumption. About 0.9 million tons of fish are caught annually in Bangladesh. Most of the marine fish (about 10 percent of the total) are dried.

Heavy losses of dried fish occur through pest infestation in two stages; (1) during drying, and (2) during storage in godowns and transportation. Fresh fish with a high moisture content is subject to heavy blow fly infes- tation (Lucilia sp.). In laboratory samples, Drubphila and house fly larvae have been found. It is understood that in the Philippines (Pablo, personal correspondence) fish is also infested with flies of the Sarcophagidae family. Fishermen have indicated that as much as 30 percent of the meat is lost due to fly infestation. There is very little done in practice to prevent flies from infesting the fish. Occasional spraying of insecticides is done very injudi- ciously with harmful results to consumers; in some cases, molasses sprinkled with insecticides are used as bait to kill the flies.

The real danger to sundried fish is beetles. They attack fish during transportation, in godowns and even at the consumer level. The most important pest in Bangladesh is the hide beetle, macularus Degeer. This species also occurs abundantly in Africa (Azab et al., 1973); Osuji, 1975; Pointel and Pham Van Sam, 1969). The copra beetle, Necrobia mfpes Degeer, and earwigs are also associated with dried fish in Bangladesh. Pablo (per- sonal correspondence) has noted the occurrence of Dermestes camivoms in dried fish in the Philippines. lnsecti- cides are seldom used in Bangladesh to control beetles. Cleanliness is maintained as much as possible but business- men pay heavily due to beetle damage; as a result, there is a tremendous price fluctuation depending on the season. LITERATURE REVIEW

A number of workers have conducted research on the biology and insecticidal control of insects in dried fish, but very few have reported on radiation disinfestation. Hinton (1955) reported that the hide beetle,D. macu- latus Degeer is a pest of great economic importance which causes extensive damage to dried products such as hides and dried fish. Alam (1971) mentioned that D. mucu&tus Degeer causes serious damage to dried fish in this country. Paul et aL, (1963) studied the life history of the beetle at constant temperature and hu- midity. He reported the incubation, larval and pupal period at 30% and 70 percent RH as 2.4,8 and 7 days res- pectively. He also recorded a life span of 87 days in male and 98 days in females. Azab et al., (1973) conducted a series of experiments to investigate the biology and factors affecting development, adult longevity and the rate of oviposition of D. muculatus Degeer at different temperatures. They found that the egg stage varied from 1-2 days at 30°c and 10-11 days at 16J°C. The larval period lasted between 24 days at 320C and 96 days at 19.4O C. The prepupal stage lasted 2-11 days according to temperature. The pupal stage averaged 5.1 days at 29.7"C and 33.1 days at 16.4 OC. Oviposition was also greatly affected (from 28-130 days) by the variation of tempe- rature and humidity. Osuji (1975) studied the development of D. macuhtus Degeer in dried fish under laboratory conditions; females laid eggs within 12 h of copulation and hatching occurred at about 48 h. After oviposition, larval development was completed in 33.5 days during which time several moults occurred. The adult emerged about 11 days after the last instar stage, irrespective of the mode of pupation.

Maclellan (1964) preserved dried fish for 6 weeks against insect attack by using a water emulsion containing 0.01 percent pyrethrin and 0.036 percent piperonylbutoxide. Lloyd and Dyte (1965) studied the susceptibility of Dermestes spp. to 15 contact insecticides. The organophosphorus compounds, dimethoate, morphothion, dicapthion, fenithrothion, fenthion, etc. were most effective in killing Dermestes spp. Dieldrin was the most effective compound used on D. maculatus Degeer but it yielded negligible mortalities with Dermestes lardarius and Dermestes ater.

Dyte et al. (1966) observed that with malathion the LD50 for larvae of D. maculatus Degeer was 10 times that for larvae of D. lardarius. However, these two species are known to have similar susceptibilities to several other organophosphorus insecticides. Kane (1967) reported that silica dusts of low bulk density showed promise for the control of Dermestesfrischii Kug, D. ater Degeer and Necrobia nrfipes Degeer infesting dried fish.

Green (1967) stated that control and subsequent protection could be achieved by dipping infested dried fish in an emulsion containing 0.02 percent pyrethrum or 0.0625 percent malathion. (1971) wheatly used several insecticides to evaluate the most promising compourds for the control of Dermestes spp. He placed the insecti- cides in order of effectiveness as dieldrin, carboxyl, Bayer 7788, Dursban and fenthion. Proctor (1972) reported that an emulsion containing 0.018 percent pyrethrin and 0.036 percent pyperonylbutoxide applied to smoke- dried fish in Zambia prevented infestation by Dermestes spp. for about 8-12 weeks. We concluded that a pyrre- thrin emulsion was economically feasible and suitable for use in Zambia. Levinson and Bashelkousky (1967) introduced antivitamins in the diet to control the hide beetle and had some success in arresting its development. Shaw and Lloyd (1969), on the other hand, reported that an Australian strain of D. maculatus, when exposed to r-BHC and reared for 15 generations on fish meal containing r-BHC, showed a gradual increase in resistance. This resistance reached 26 times that of the susceptible strain by the F14 generation. Toye (1970) found that heating dried fish in an oven at 60°C for 30 min at fourday intervals, in a simple charcoal-fired oven, and storage in metal containers would control the pests. Mushi and Chiang (1974) observed that dried fish containing 13 per- cent, or more, salt could prevent the growth of insects at all developmental stages.

Daget (1966) used ionizing radiation for the disinfestation of dried fish in sealed containers; this resulted in death or sterilization of the insects without any harmful effects to the products. Boisot and Gauzit (1966) reported that 30 percent by weight of the fish was destroyed by insect infestation and that none of the conven- tional methods of control was successful. They used various doses of radiation and the dose necessary for sterili- zation was estimated as between 15 000 and 40 000 rad; no organoleptic changes were observed at 50 000 rad. They also studied the taste and nutritional value of the dried and smoked fish after exposing them to radiation doses ranging from 20 000 to 50 000 rad; no adverse effects were reported. Pointel and Pham Van Sam (1969) used irradiation with doses from 10 to 30 krad to find out the sterilization dose for D. maculatus Degeer. None of the treated stages gave any progeny and their life span was shortened.

DISINFESTATION OF GONIA, CARP AGAINST EARWIGS

Earwigs are also a,problem in dried fish and it was decided toestablish the effects of irradiation treat- ment on dried gonia (Labeo gonius). With some difficulty a mixed population of these insects was built up in the fish and no attempt was made to separate earwigs into age groups. The fish were exposed to gamma rays from a cobalt 60 source at doses-of 10,20 and 30 krad. Each sample had about 50 g of dried fish and each treatment was replicated four times. After 12 days, the number of insects, both dead and alive, were counted (Table 1.). There was considerable damage in the controls but almost none in any of the irradiated samples. The number of dead insects in the control was significantly fewer than in other treatments; live .inseas were found only in the con- trols. It can be seen that the fish were completely disinfested by these doses of radiation. Further observations were made after another 3 weeks but there were nb live insects and none of the sarnples was damaged.

Table 1

Effect of irradiation on earwigs associated with dried fish, Labeo gonius

I I Irradiation dose (krad) Sample no. (number of dead Iarvae)

0 10 20 30

4 3 (7) -- 7 8 7

Average 2.75" * 6 .ob 7.79 8.50~

Number in parentheses = numberof alive larvae 2 * Number designated by unlike letters are significant Least significant difference between treatments (LSD) at 5% level = 3-09 Least significant difference between treatments (LSD) at 1%level = 4.75

DISINFESTATION OF DRIED MACKEREL

Mackerel is an important fish in the Bay of Bengal. It is estimated that about 15 percent of a total catch of 80 000 tons of marine fish is mackerel, most of which is dried. It is one of the most expensive dried fish. Experi- ments were conducted to determine the disinfesting dose for eggs, larvae, pupae and adults of the hide beetle, D. maculatus Degeer; each treatment was replicated five times. Materids and methods

Laboratoryreared insects, gown on mackerel, were used for all the experiments. Dried mackerel were heated in an oven for 24 h at 50°C. For convenience, Petri dishes (9 cm diameter) and small inseftrearingjars (5 x 8 x 15 cm) were used to maintain the experimental insect population. The Petri dishes were covered with tissue paper for aeration before replacing the lid; rearing jars were covered with muslin which was secured with rubber bands.

Hide beetles lay eggs in crevices and stick them to dried fish. When the eggs were separated from their substratum, a large number became damaged. Therefore, an indirect method was used for the irradiation of eggs. Adults were released on dried mackerel and after 48 b, 40 g of dried fish were taken for each replicate. The dried fish with eggs (048 hour old) were exposed to 0 (control), 25, 5, 7.5, 10 and 15 krad of gamma rays.

Larvae

Last instar larvae were separated from stock cultures; these were 25-28 days old and a few had started to pupate. Fifty grammes of dried mackerel were placed in a rearing jar and 20 larvae were added. These were irra- diated at 0, 2.5, 5, 7.5, 10, 15 and 20 krad. Observations were made every other day for modity and feeding. Fresh food, which was irradiated at the same time as the larvae, was added only when necessary.

Pupae

The pupae selected for irradiation were 2-4 days old; 20 pupae were added to 50 g of dried fish. The samples were exposed to 0, 5, 10, 15 and 20 krad and then the pupae were placed in Petri dishes. Dsily observa- tions were made for mortality of pupae, adult emergence and adult mortality, As the adults emerged they were transferred to small rearing jars containing treated food.

Adults

Adults, 6-7 days old, were set up similarly and were exposed to 0, 5, 10, 15,20, 30 and 40 krad. The ir- radiated adults were housed in rearing jars and observations were made on alternate days for feeding, mortality and production of F 1 progeny if any.

Sterility doses

The pupae were separated from the rearing media and each was placed in a separate vial to ensure emerg- ence of unmated adults. Males of 2-3 days old were exposed to 1, 1.5,s and 10krad of gamma radiation. After treatment, 5 males were combined with 5 females in rearing jars (15 x 8 x 5 cm) to determine the doses for sterility.

Results

The observations were made after 10 days; the fish were broken up carefully and the number of he either dead or alive was counted (Table 2). All the larvae in the treated samples were dead, although there were some cast skins. There was normal feeding in the controls, whereas feeding (as determined from the damaged fish) was reduced increasingly with increased radiation doses. At the time of observation, all larvae in the controls were second and third instars; larvae in the treated samples died in first and second instars. This was established by the number of cast skins, which at 2.5, 5, 7, 10 and 15 krad, were 21.4, 166, 10.2, 4.8 and 3.4 respectively com- pared to 40 in the control. Table 2 Effect of irradiation on 0 to 48-h old eggs of Dermestes manclatus Degeer 10 days after treatment

2 Irradiation dose (krad) Sample no. (number larvae dead or alive)

O* 2.5 5 75 10 15

1 20 45 46 10 15 24

Average 1 30.8 4024 49 -6 31.24 17.24 16.6 1 * All larvae in control were alive; none was alive in the irradiated groups Larvae

Results of larval mortality are shown in Table 3 and Fig. 1. Larvae are susceptible to irradiation; a dose as low as 5 krad gave 90 percent mortality compared to 26 percent in the control. The number of dead larvae was highly significant in treated insects compared to the control. Feeding after a dose of 10 krad and above almost stopped. Fig. 1 also shows that there was a peak of mortality on the eighth day after treatment. Although the insects exposed to the higher doses continued to live, they were moribund and could be considered as dead. None of the irradiated insects pupated. Pupation was 74 percent in the controls and the adults which emerged subsequently produced normal F 1 progeny.

Table 3 Effect of irradiation on last instar larvae of D. maculatus 20 days after treatment Irradiation dose (krad) Sample no. (number of dead larvae)

0 2 -5 5 7.5 10 15 20

1 8 16 18 8 15 18 20

* Numbers designated by unlike letters are significant

LSD at 5% level = 431 LSD at 1% level = 6.12 Pupae

Radiation effects on pupae are summarized in Table 4; unlike larvae, irradiation did not drastically check adult emergence. Emergence of adults was 89 percent in the control and 72 percent in those exposed to 20 krad. The percentages of deformed adults from pupae treated at 0, 5, 10, 15 and 20 krad were 0, 17, 32,21 and 36 respectively. There was a significant difference between the highest and the lowest irradiated treatment. All adults at 20 krad were deformed. Normal adults from irradiated pupae were allowed to mate but no progeny was obtained. Control adults produced normal progeny.

Adults

Mortalities in adults were recorded for 24 days. Dose response curves are shown in Fig. 2; it is evident that radiation drastically affected the longevity of adults. There are two peaks: the first was due to higher doses of radiation and the second to lower doses of radiation. Table 5 shows that after 14 days, over 90 percent mortality was obtained at a dose of 15 krad compared to 18 percent in the controls. Insects at 5 and 10 krad initially showed normal feeding but, afterwards, feeding was much reduced; adults at doses of 15 and 20 krad showed little feeding from the very beginning. Adults subjected to 30 and 40 krad seemed to get a shock after irradiation but recovered after a couple of days. Feeding was almost stopped at 30 krad and there was no sign of feeding at 40 krad. However, at higher doses, movement of insects was greatly affected. They continued to live although moribund. Control insects were all normal.

Sterility doses

It was observed that 50 percent sterility could be induced in males by a dose of 1 krad. A dose of 5 krad could be considered an effective dose for inducing almost 100 percent sterility.

Table 4

Effect of irradiation on 2-4 day-old pupae of D. maculatus

Irradiation dose Number of adults Numbej of adults Number of adults (krad ) emerged deformed dead after 20 days

Control 17.8 0 2.4" 5 1424 3.4a* 13.6~ 10 15.00 6.4bc 14.4~ 15 11.8 4~4~~ llsb 20 14.40 724C 14.40~

LSD 5% 3.43 2.34 421 LSD' 1% 5.03 3.43 6.12

Numbers designated by unlike letters are significant -Control (P -pupation, A - adult) --2.5 Kmd 5 Krad -7.5 Krad ...... 10 Krad ---c- 15 Krad

Figure 1 - MorrPlity of the lut instu larvae of Dennestes moculatus after being expod to gunmr.ixys

50-

40- +15 Krad I -30 Krad ,g -- 40 Krad i 30- f \ 5 0' 20-

lo-

Day. Figure 2 - Mortality of the dubof D. mcrnJcltus after exposure to gammaqays 317 Table 5

-Percentage mortality of irradiated 6-7 day-old adults of D. maculatus 14 days after treatment

Irradiation dose (had)

Sample no. 0 5 10 15 20 30 40

25 65 85 100 95 95 100 0 80 75 85 90 100 100 15 95 90 90 85 100 100

* Numbers designated by unlike letters are significant LSD at 5% level = 9.96 LSD at 1% level = 14.47

PACKAGING SUDIES ON DRIED FISH

Materials and methods

Dried mackerel infested with hrgc numbers of insects were wdfor this urperiment but, to ensure heavy infestation, a number of insects in different developmental stages were added to the dried fish and left for48 h. Some fish were also heated in an oven for 48 h at 60°C. Samples or 250 g were packed in polythellr pouches, 2 5 x 16 cm, and sealed.

Two controls were used; (1) untreated control and (2) heat-tteated.controI. Videwminations were made for insect damage, insect mortality, development of mould and the condition of the polythene pouches, particu- larly insect punctures. Thc following experiments were carried out:

Experiment 1

The pouches (SO p thick) were exposed to 10,20,50 and 100 krad of gamma rays; two controls were also maintained. The moisture content of the heated control was 1.3 percent; all the other samples contained 18 .per- cent. AU pouches were maintained in an incubator at 29 i10C and 70 f 5 percent dative humidity. The experi- ment was replicated four times.

Experiment 2

The pouches (75 11 thick) were exposed to 50, 100,200 and 400 krrrd with two controls as in Experiment 1. Moisture content in the hcatacated control was 13 percent; while the other samples had 20 percent moisture. The experiment was replicated five times. Experiment 3

Dried mackerel was kept in an oven at about 60°C for 24 h. The moisture content was 13 percent. Pouches were made from polphene of thickness 50, 75 and 100 u. The same doses of radiation were given as in the earlier experiment with similar controls. Five replicates for each thickness of polythene were made.

Results and observations

Experiment 1

In the untreated control samples normal feeding of insects continued. After 45 days, fungus started to develop in all but one replicate. After 65 days, all packages were completely spoiled due to the heavy growth of fungus and insect damage; living insects, in all developmental stages, were observed. Final observations were made at 100 days; a few insects were still alive and one package was punctured by insects. The heat-treated control was very good from the beginning. At the end of experiment no insect damage of fungal development was noticed in any of the pouches. The pouch were in excellent condition. At 10 krad the condition of pouches uras good up to 45 days with only slight insect damage. Very few living insects were seen and they were moribund. Slight development of mould was noticed after 65 days but all the insects were dead. In the final examination, after 100 days, 75 percent of the samples were totally spoiled due to mould; very slight insea feeding was noticed. At 20 krad no apparent insect damage was noticed except in one pouch 65 days after treatment. Fungus had developed in all replicates after 45 days' storage but spoilage was considered moderate after 65 days of storage. At the end of the experiment, 50 percent of the product was totally spoiled due to fungal growth and slight insect damage was observed in one replicate. At 50 krad treatment, the condition of the product was good for 65 days. No live insects were seen 15 days after the treatment and there was no insect damage in any of the replicates though the product had some indi- cation of fungal development. After 100 days, 75 percent of the product was completely spoiled due to fungus. Condition of the packages however was very good; none was punctured by the pests.

Experiment 2

After 2 weeks insects in all developmental stages were present in the control. There were also Necrobia rufi- pes. No dead insects were found in the pouches. There was enormous feeding by the insects but no development of fungus and the pouches were not punctured. During the same period, dead insects were observed at 50, 100 and 200 krad with no development of fungus. Slight damage by insects was noticed in one replicate each at doses of 50 and 100 had. The condition of 400 krad dose was very good; there was no indication of insect damage or fungal development. The pouches in all treatments were in good condition. The heat-treated control was similar to the 400 krad sample. Development of mould was noticed in all treatments after 4 weeks and fungal development continued in all except the heat-treated control. The experiment was stopped at 90 days by which time the control was totally destroyed due to insect feeding and fungal development and a few insects, in developmental stages, were still alive. All other treatments, except the heat-treated control, had heavy fungal growth; it was noticed that 40-60 percent of the product was totally spoiled. However the heat-treated control was in very good condition with no insect puncture. Dead insects were found in all irradiated samples and species of D. maculatus Degeer and N. rufipes Degeer were present.

Experiment 3

Observations were made over 45 days. There was heavy insect damage in the control. No development of fungus or insect damage to the fish was noticed in any of the other treatments. Insects in the irradiated treat- ments appeared to be dead. The condition of the pouches was very good in all treatments except the control. DISCUSSION

Disinfestation experiments withLabco ~oniuswere successful. Although we could not establish whether earwigs were the primary pests in heavily infested godowns containing rejected dried fish, their presence contri- buted to the damage of fish. After one month of experiment, all earwigs disappeared indicating that such medium was not suitable for their growth.

Eggs of D. maculatus Degeer were most susceptible to radiation. Even the low dose of 2.5 krad prevented the eggs from developing into fully mature larvae. A dose of 5 krad controlled 90 percent of larvae within 2 weeks. Feeding was stopped at 10 krad and above; a dose higher than 10 krad should be used to control larval infestation. As there was no adult emwgence in any of the treated larvae, further infestation could not oc ur.

Irradiatioq at the pupal stage did not check adult emergence even at lower doses (Table 4). There was no significant difference between treatments except between the control and 15 krad. It was observed that as the dose increased the percentage of deformities in adults also increased. All the adults which emerged from 20 krad were deformed. Normal adults from irradiated pupae were all sterile. Feeding of these adults was also drastically reduced; as such these could be considered harmless in the dried fish. From the dose response curves of Fig. 2, it appears that longevity of irradiated adults was much affected thereby curtailing their period of infestation. A dose of 15 krad killed 90 percent with 2 weeks; to check spoilage instantaneously, a dose of 30 krad was needed. It also appears that a dose of 20 krad could be considered for practical use as the feeding of adults in this treat- ment was very negligible.

It is apparent from these experiments and from other studies that earwigs and hide beetles can be elimi- nated by the use of gamma rays. The results of Daget (1966) Boisot and Gauzit (1966) and Pointel and Pham Van Sam (1969) agree with the present experiments. Beetles of all stages can be disinfested at 20-30 had. This dose falls well within the permissible dose of radiation used in the disinfestation of cereals and cereal products in many countries.

From the three packaging experiments it was found that a dose of 50 krad and above would kill all insects. It was fungus which caused much of the spoilage of dried fish. In practice, jute bags are used for the transporta- tion of dried fish. These bags allow the passage of air and the problcm of mould growth is obviated. Quality of thc heat-treated control remained very good. For the radiation doses as used in this experiment, the fish should be thoroughly dried for successful disinfestation.

All the thickness of polythene used were resistant to insects. There were however a few punctures in pouches of 50 u thickness; 75 J,I thickness would seem to be adequate for practical use. In Bangladesh pofy- thene is considered to be a good packaging material since it is abundantly produced and readily available. It is also comparatively cheap.

ACKNOWLEDGEMENT

This study was partly supported by the 1nterna:ional Atomic Energy Agency, Vienna under a Research Contract.

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