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The germ-free culture of desert locusts, Schistocerca gregaria
0022-1910/85 $3.00 + 0.00 Copyright CC:1985 Pergamon Press Ltd
Physiol. Vol. 31. No. 6. pp. 477485, 1985 Printed in Great Britain. All rights reserved
J. Insect
THE GERM-FREE
CULTURE
SCHISTOCERCA A. K. School of Biological
CHARNLEY,
Sciences,
University
OF DESERT
LOCUSTS,
GREGARIA
J. HUNT
of Bath,
and R. J. Claverton
DILLON
Down,
(Received 16 August 1984: revised 7 November
Bath BA2 7AY, U.K.
1984)
Abstract-A technique is described for rearing germ-free desert locusts. Axenic insects appeared to develop and reproduce normally, therefore it is concluded that the gut bacterial flora does not contribute significantly to locust nutrition. However, it is suggested that the gut flora may benefit its host by contributing to the locust’s defences against attack by pathogenic microorganisms. The results of morphometric measurements are discussed in terms of the bacterial origin of the so called phase pheromone. locustol. No evidence was found to support the hypothesis that microbial activity restricts gut carbohydrase. Key Word Index: Locust,
Schistocerca. bacteria,
axenic
MATERIALS
INTRODUCTION
In a previous publication we reported an abundant bacterial flora in the gut of Schistocerca gregariu (Hunt and Charnley, 1981). In particular a large population of Enterobacteriaceae was found with numbers increasing posteriorly from foregut to rectal sac. Whereas bacteria in the anterior regions of the gut were restricted to the lumen and the inside of the peritrophic membrane, bacteria in the hindgut were intimately associated with the cuticular lining particularly at the bottom of the folds in the ileum and between adjacent rectal pads. Little is known of the relationship between the desert locust and its gut bacterial flora. Bacteria have been implicated in the production of the so called gregarization pheromone, locustol. in a related species, Locusta migratoria (Nolte, 1977), and Strebler (1978) has suggested that microbial waste toxins may limit carbohydrase activity in the gut of S. gregaria. However, the evidence for these observations is largely circumstantial. The object of the present work was to devise a technique for culturing S. gregaria axenically and to effect a preliminary physiological characterization of germ-free insects with particular regard to the work of Nolte (1977) and Strebler (1978). The protozoan parasites Mulameba locustae and Gregarina garnhami are widespread if not universal problems in laboratory cultures (Canning, 1956; Henry, 1968; Tobe and Pratt, 1975). Therefore comparisons have been made between germ-free, conventional and parasite-free locusts. Many species of insect have been reared axenically on synthetic diets in conjunction with nutritional studies (House, 1972). However, the taxonomic distribution of the species reared axenically is biased towards the orders of Coleoptera, Diptera and Lepidoptera; there appear to have been few attempts to culture Orthoptera in this way. In addition, few species of insect have been reared on prospectively near-normal natural food with axenicity the matter of central interest rather than nutrition. 477
AND METHODS
Colonies of desert locusts, Schistocerca gregaria and migratory locusts, Locusta migratoria, were maintained according to the protocol of HunterJones (1966). Axenic desert locusts were reared in flexible plastic (PVC) isolators (see Fig. l), a method that has been employed extensively for producing germ-free vertebrates (see Coates and Fuller, 1977). Axenic insects (free from any detectable form of microbial life, no attempt was made to extract viruses) were compared with two groups of controls: conventional insects (taken from the stock colony) that had a “normal” bacterial flora and were infected with M. locustae and G. garnhami, and parasite-free insects that had only a bacterial flora. The last named were produced by raising axenic hoppers (see below) in isolators without bacterial filters in rooms which had not previously housed locusts. Populations of the three treatments were started off with 100 lst-instar larvae. Experiments were performed on 1Cday old adults and unless otherwise stated equal numbers of males and females were used. Sterilization
of equipment
Equipment was sterilized initially by irradiation, steam (121°C) or chemically using 2% peracetic acid (Laporte Inds.) as appropriate. During the construction of the isolator, the pvc unit and all associated items and furnishings were thoroughly sprayed with peracetic acid. A period of 2 days then elapsed before the axenic 1st-instar larvae were introduced, to allow the isolator to dry out, degradation of the sterilant and the removal of the volatile breakdown product acetic acid. Production
qf axenic
lst-instar
larvae
Seven to eight day old locust eggs were removed from egg tubes and carefully washed with distilled water to remove sand particles. Misshapen or discoloured eggs were discarded as potential reservoirs of bacteria. Batches of 4&50 eggs were surface sterilised by placing in 2”/, peracetic acid for 20 min. washed in
478
A. K. CHARNLEY PI (11.
sterile distilled water and then transferred aseptically to individual sterile tubes (50 x 10 mm) containing nutrient agar slopes (Oxoid). Eggs were incubated at 32 C to allow completion of development. The agar slopes were examined daily with the aid of a microscope so that contaminated eggs could be detected and discarded. Upon hatching the hoppers were retained in the tubes for a further 2 days during which time they ate the agar and voided faecal pellets. If the agar was still apparently free from microbial growth then the insect was regarded as axenic. The tubes containing the hoppers were then placed in the entry port (see Fig. 1) and sprayed with 2Y; peracetic acid, after 60min they were transferred into the isolator and released into the cage. Maintenunce of experimental insects All three treatments were maintained at a temperature of 32 f 3.C, under a 12 h light, 12 h dark photoperiodic regime. Control of cage humidity was not possible because of the destabilizing effect of daily additions of moist grass. In practice restricting feeding to early morning and late afternoon produced a diurnal relative humidity cycle that was constant between treatments (range 50-75:,;). All experimental groups were fed the same diet (unless otherwise stated); equal proportions of autoclaved ( 121°C for 30min) and vacuum packed y-irradiated (5 mega rads (IPI, Swindon)) freeze-dried grass and bran. Dry grass was rewetted with sterile water prior to feeding. Breeding usenic insects Aluminium tubes containing damp sand (HunterJones, 1966) were placed under the holes in the false floor of the insect cage. After l-2 egg pods had been laid the tube was capped, removed from the cage and kept in the isolator until the eggs were ready to hatch. At this time the tube was transferred to a 2.5 1 jar
within the isolator, where the hoppers were kept until the 3rd instar when they were transferred to the cage. Microbiological control oj’germ+ee
isolators
Clearly a reliable system of sterility testing is crucial for the validity of the operation. Procedures have been described by many authors (e.g. JayneWilliams and Hewitt, 1972; Benschoter and Wren, 1972). The protocol adopted in the present work was based on the recommendations of Fuller (1968). The germ-free status of insects in the isolators was confirmed after each ecdysis by placing samples of faecal pellets and damp swabs of cage and isolator into tubes of nutrient broth stocked inside the isolator. Tubes were examined daily (for 4 days) for signs of bacterial and fungal growth. Two days before the insects were removed for experimentation more extensive tests were performed. One animal was killed. its gut removed and the contents placed in a tube of nutrient broth; the rest of the cadaver was placed in a second tube of broth and both tubes were examined daily as before. Samples of faecal pellets were added to tubes of nutrient and thioglycollate broth (Oxoid, CM23). The nutrient broth tubes were incubated aerobically and the thioglycollate tubes anaerobically (Gas Generating Kit, Oxoid) at 32OC for 7 days. A sample from each tube was streaked onto nutrient and blood agars (Hunt and Charnley, 1981) and these were also incubated at 32°C for 7 days. Furthermore, the peritrophic membranes were removed from 20 faecal pellets, Gram stained (Ollett, 1947) and examined microscopically. Animals used for experiments were routinely examined for the presence of parasites including Malameba locustae and Gregurinu garnhami. Insects were considered to be Malameba-free if Malpighian tubules, gut and faecal pellets of mature adults were devoid of cysts when viewed in squash preparations and during light and scanning electron microscopy (Hunt and Charnley, 1981). No attempt was made to isolate any viruses present in the animals. If no microorganisms or parasites were detected by any of the tests the animals were considered to be axenic. Isolutiorl. enumeration und identt;fication of’ bucteriu The techniques employed described (Hunt and Charnley.
were as 1981).
previously
Lipid. curbolydrate and protein meusurements Blood was collected in 5 ~1 Drummond microcaps from a small puncture in the arthrodial membrane at the base of a hind leg. Total lipid concentration in samples was quantified using the phosphovanillin method as described by Ziillner and Kirsch (1962). Analysis of carbohydrate was performed with the anthrone method of Roe (1955). Protein was estimated by the micro-biuret method of Itzhaki and Gill ( 1964). Fig. I. Flexible pvc isolator system used to rear germ-free desert locusts. Filter-sterilized air was continually pumped through the isolator to maintain a positive pressure, thus reducing the possibility of contamination due to inward leakage of non-sterile air. Manipulations within the isolator were carried out using the gloved-sleeves. A, entry port: B, plastic isolator (position of the gloved-sleeves depicted by dashed circles): C. locust cage; D. outlet filter; E. inlet filter; G. pump; H. heater.
Assu~s fiw digestive enzyme acthit? Guts were dissected out in ice-cold saline (Maddrell and Klunsuwan, 1973). As required, midgut (tissue or contents of the lumen) and hindgut (tissue and contents of the lumen) were homogenised in distilled water, then centrifuged for 15 min at 2000 rpm. Supernatants were divided into 0.5 ml ali-
Germ-free locusts Table I. Vitamin supplement used in the development of the axenic diet 25 rg/g 25 I(& 25 rg/g 50 pg/g 100 fig/g 25 pg/g 125 pg/g 1 pg/g 250 @g/g 100 p&g 1.4 mg/g
Thiamine Folk acid Riboflavin Pantothenic acid Nicotinic acid Pyridoxine Choline chloride Biotin Inositol Ascorbic acid Carotene
“Single” vitamin supplement. quantities expressed per g of diet (from Cavanagh, 1963).
quots and kept deep frozen at -2O’C until required. The activities of tl- and fl-glucosidase were determined by the estimation of p nitrophenol liberated from the corresponding p nitrophenyl glycoside. Reaction mixtures of 2 ml 0.1 M McIlvane’s buffer pH 5.4 (Dawson et al., 1969) 0.5 ml of the substrate and 0.5 ml of the homogenate were incubated at 28°C for 15 min. The reaction was terminated by the addition of 3 ml of NH,OH (Aizawa, 1939) and the absorbance read at 405 nm. The activity of p-l,3 glucanase was determined by estimating the reducing sugar (glucose) released from laminarin using Nelson’s (1944) modification of the Somogyi method, observing the precautions outlined by Marais et al. (1966). Reaction mixtures of 2 ml of McIlvane’s buffer pH 5.3. 2 ml of 0.01% laminarin solution and 0.1 ml of homogenate were incubated for 1 h at 28°C. The reaction was stopped by adding 2ml of copper reagent (Nelson, 1944) to 2 ml of the incubate. The mixture was heated in capped tubes for 25 min at 100°C then cooled to 21°C. Two ml of arsenomolybdate reagent (Nelson, 1944) was added to the tube and mixed on a Vortex mixer until effervescence ceased. After 2 h colour development was complete and stable (Marais et al., 1966) and absorbance of appropriately diluted samples was read at 660nm. Morphometric measurements The length of a hind femur (F) and greatest width of the head (C), were measured to the nearest 0.01 mm using vernier calipers and a micrometer screw gauge respectively. The F/C ratio was then calculated (Dirsch, 1953). Statistics Statistical comparisons of data were performed using Student’s t-test (Sokal and Rohlf, 1969).
RESULTS
Microbiological status qf the guts of axenic, parasitefree and conventional locusts Locusts reared axenically were found to be free from microbial contamination when faecal pellets, swabs, excised guts and cadavers were assessed by the methods described in the previous section. Further-
479
more their guts and Malpighian tubules were free from the parasites M. locustae and G. garnhami. Animals reared in the absence of parasites had a mean of 2.2 x lo6 bacteria (Enterobacteriaceae)/ml (n = 11) in the hindgut. All conventional insects were infected with M. locustae and G. garnhami and had a hindgut bacterial density of 2.1 x 10” bacteria (Enterobacteriaceae)/ml (n = 56). Development of the axenic diet Dadd has defined the majority of the nutritional requirements for the growth and development of S. gregariu (Dadd, 1963). However, neither he nor Cavanagh (1963) were able to produce a complete synthetic diet; a small daily supplement of fresh grass was necessary to eliminate low fertility of eggs. In the light of this it was decided to use the normal laboratory diet of grass and bran as the basis of the axenic diet. Preliminary experiments showed that vacuumpacked freeze dried bran and grass (rehydrated before feeding) was efficacious, confirming the observations of Louveaux et al. (1980). Vacuum-packed freeze-dried grass and bran was sterilized by y-irradiation (5 mega rads) and batches of 100 newly hatched conventional L. migratoria and S. gregaria larvae raised on the diet to adult (diet 2) Only 2% of Locusta and 9% Schistocerca developed to adult, compared with 29 and 26% respectively of the controls reared on untreated grass and bran (diet 1). The failure of the experimental diet would seem to be due primarily to a lack of vitamins, as a vitamin supplement (Table 1) vastly increased the proportion of Locusta surviving to adult (22%, diet 3). Previous work has shown that B vitamins in particular are susceptible to irradiation (Ley et al., 1969) although the exclusion of oxygen and water, as practised here, provides some protection (Coates et al., 1969). A second experiment was performed with Schistocerca in which irradiated food was supplemented with an equal quantity of autoclaved grass (diet 6). This proved to be a better diet (10% ecdysed to adult) than the unsupplemented irradiated diet (5”,/, ecdysed to adult, diet 2). However, once again the addition of vitamins to the bran either before (14”/, ecdysed to adult, diet 5) or after (16% ecdysed to adult, diet 4) irradiation proved optimal. In both cases 1Cday old adult females were heavier than those reared on diets 1 and 2 and a greater proportion of the insects ecdysed to adult. Double quantities of vitamins were used when the supplement was added to the bran prior to sterilization, in an attempt to counter the deleterious effects of y-irradiation. This seems to have been effective because there was little difference between the efficacies of diet 4 (single quantities of vitamins added to the bran after irradiation) and diet 5 (double quantities of vitamins added to the bran before irradiation). Finally insects were reared on a mixture of irradiated bran and grass + autoclaved bran and grass (diet 7). This diet proved to be at least as nutritious as those supplemented with vitamins, in addition similar proportions of desert locust populations reared on diet 7 and on the normal diet ecdysed to adult (25 and 26% respectively), consequently diet 7
A. K.
CHARNLEY
adult population that was similar to parasite-free and conventional insects (Fig. 2). Populations of axenic locusts took twice as long as groups of parasite-free locusts to complete the metamorphic ecdysis (Fig. 2). An even more marked desynchronisation of development occurred amongst conventional insects. The mean wet weight of 1Cday old female axenic adult locusts was not significantly different from that of comparable parasite-free animals, though male axenics were in fact significantly heavier than male parasite-frees (Table 2). Males and females of both groups were significantly heavier than their conventional counter-parts. Fat body weights of male locusts followed a similar pattern to the female whole body weights viz. although there was no significant difference between axenic and parasite-free insects, both had much more fat body than the conventional insects (Table 2).
60 2
10
20
30
40
Days
Fig. 2. The development and pattern of mortality of axenic. parasite-free and conventional locusts. The graph represents the results from a typical experiment. Horizontal dotted lines indicate the spread of the metamorphic ecdysis. Abscissa, time in days after egg hatch Ordinate, O0 of the population alive after a given ecdysis (each point represents an ecdysis). l PO Axenic; n -m Parasite-free; A-A Conventional.
was adopted as the experimental of axenic insects. Growth and detlelopment
et uI
Breeding axenic locusts On one occasion 10 axenic adults (5 females and 5 males) were kept for breeding. Over a period of I7 days from the onset of laying I2 egg pods were produced. The details are given in Table 3: they compare favourably with data for conventional locusts reared on grass and bran by Hamilton (1955). There is considerable variation in the statistics of desert locust egg production reported in the literature. In contrast to Hamilton (1955), and the present work. Cavanagh (1963) found ca. 87 eggs/pod and 26 days to production of 1st pod. However, the viability of eggs laid by the axenic locusts (>70% hatch) was within the range observed by Cavanagh (70.&98.2”,). First generation axenic hoppers were successfully reared to adult and their parents were still alive after 120 days of adult life.
diet for the rearing
of‘ axenic locusts
the course of 4 experiments. each involving 100 insects per treatment. 29% of axenic locusts successfully developed to adult, compared with 279, of parasite-free and 25”,/, of conventional insects. Since there was little difference between axenic and parasite-free treatments it may be concluded that S. gregaria does not need a bacterial flora to successfully complete larval development. However, differences were observed in the pattern of mortality of the three experimental groups during development (Fig. 2). There was a high mortality rate amongst the early instars of parasite-free and conventional animals, only 50% of the initial population reached 3rd instar. The mortality rate then slowed down so that 2&40x achieved adulthood. In contrast to the other two treatments the axenic animals had a much reduced mortality in the early instars (80% survival to the 3rd instar). However, increased mortality during the later instars resulted in a final During
Concentration haemolJwiph
qf’lipid, protein and carbohydrate
The concentration of key macromolecules in the haemolymph of locusts are known to vary with the physiological state of the animal (Goldsworthy, 1969: Hill and Goldsworthy, 1970; Gordon and Webster, 1971). In the light of this we decided to look at the titres of protein, lipid and carbohydrate in axenic locusts to see if there were any indications of metabolic disruption. The results are recorded in Table 4. There were no significant differences between axenic and parasite-free insects with any of the parameters measured. However, conventional insects
Table 2. Whole insect and fat body weights of axenic. parasite
free and conventional
Weight of insect (g) Male Rearing
conditions
Axenic (Ax) Parasite-free (PF) Conventional (C) Significant
F%%TI& N
.r
SE
N
y
SE
N
2.15 I .96
0.05 0.07 0.03
44 9 42
3.31 3.03 1.33
0.09 0.15 0.12
37 12 18
87.0 19.9 5.3
16.9 15.4 2.01
10 9 7
Ax v C. PF Y C Ax Y PF Others
P
Male
SE
Not significant ‘P = 0.05-0.02.
locusts
Wt fat body (mg)
.I
1.66
< 0.001
in the
Ax v C. PF v C
Ax v C, PF v C
Ax v PF
Ax v PF
Germ-free locusts Table 3. Comparison
of axenic and conventional
Hopper period (days) Age of adult (days) when 1st pod produced Adult life (days) Eggs/Pod Incubation period (days)
There was no significant difference between conventional and axenic insects with any of the three enzymes in either tissue or contents of the midgut. Interestingly hindgut a-glucosidase activity was significantly greater in conventional animals than in animals reared axenically or parasite-free. The activity was directly related to the concentration of bacteria (Enterobacteriaceae) in the hindgut; animals which had the most bacteria also possessed the highest a-glucosidase activity (Table 5B). The opposite to that expected on the basis of Strebler’s hypothesis.
locust production
Axenic
Conventional (Hamilton, 1955)
28
28.6 21 68.3 40 14.7
30 >l?O 42” 14
‘> 709/, of the eggs hatched.
had a significantly lower lipid concentration axenic and a significantly higher carbohydrate centration than the parasite-free insects. Protein in the haemolymph of the three treatments similar. Carhohydrase
activity
Morphometric
than contitres were
in the gut
of lipid, protein
Lipid
measurements
In a series of papers Nolte and his colleagues (Nolte, 1963; Nolte, 1977; Nolte et al., 1970; Nolte et al., 1973) claimed to show that crowded locusts produced a pheromone (locustol) which affected phase polymorphism, making solitary locusts more like the gregarious form. The pheromone was nonspecific and was produced by Schistocerca gregaria, Locusta migratoria and Locustana pardalina (Nolte, 1973). It was demonstrated that antibiotic-treated, crowded Locusta exhibited solitary characteristics (Nolte, 1977) and the hypothesis was made that locust01 was produced by bacteria in the crop and was excreted with the faeces. Unfortunately, this work has received serious criticism. In particular a rigorous study by Dearn (1974a,b) failed to confirm the validity of Nolte’s principal (but not only) criterion of phase change, viz. an increase (gregarious)
Strebler (1978) found enhanced carbohydrase activity in the guts of desert locusts which had been either starved or fed antibiotics. He suggested that under normal conditions microbial waste products and toxins limited enzyme activity. We endeavoured to seek support for Strebler’s hypothesis by assaying for tl- and b-glucosidase and p 1:3 glucanase activities in the midguts and hindguts of locusts of the three treatments (see Tables 5A and B). Considerably more activity of all 3 enzymes was found in the lumen than in the tissue of the midgut. Table 4. Concentration
481
and carbohydrate Protein
(pg/ltl)
in the blood
of S. gregaria Carbohydrate
@g/PI)
@g/PI)
R
SE
N
R
SE
N
x
SE
N
Axenic (Ax) Parasite-free (PF) Conventional (C)
11.29 10.70 8.25
I.2 1.4 0.36
20 8 30
31.4 43.3 39.5
2.5 3.2 3.1
19 8 20
20.0 18.2 23.1
I .4
IO 7 18
Significant Not significant
Ax v C (P =0.02-0.01) Ax v PF, PF v C
Rearing
conditions
activity
in the locust gut Lumen activity
Tissue activity A. Midgut r-Glucosidase” Conventional Axenic 8.Glucosidase” Conventional Axenic p-1 :3 Glucanaseb Conventional Axenic Comparisons
between
treatment
R
SE
N
R
SE
N
4.39 2.2
0.73 0.67
12 I2
35.49 29.87
7.1 3.29
12 12
0.91 1.5
0.13 0.33
12 I2
13.21 13.18
0.82 1.28
12 12
0.98 0.7
0.13 0.07
12 12
3.27 3.13
0.24 0.09
12 12
not significant. Tissue + lumen activity
B. Hindgut z-Glucosidase” Axenic (Ax) Parasite-free (PF) Conventional (C)
PF v C (P = O.OW.02) Ax v PF, Ax v C
Ax v PF, Ax v C. PF v C
Table 5. Carbohydrase
2.3 1.1
‘Bacterial
density/ml
x 10’
R
SE
N
R
SE
N
0.07 0.29 I .49
0.01 0.07 0.23
10 IO 10
0 0.22 21
0 0.02 3.0
10 IO 56
All treatments significantly different from each other, for both enzyme activity and bacterial at P < 0.001. “Activity expressed as pmol p-nitrophenol released/gut region/l5 min. bActivity expressed as mg glucose released/midgut region/hour. ‘Enterobacteriaceae (Hunt and Charnley, 1981).
-
density,
482
A. K.
CHARNLEY et cd.
Table 6. Morpbometrlc
measurements
of adult locusts
FC .Y
SE
N
Axrnic (Ax) PamsIte-free (PF) Conventional (C)
3.50 3 39 3.2x
0.02 0.02 0 07
39 31 30
50 66 Xl
Muk Awnic (Ax) Parasite-free (PF) Convrntional (0
3 43 3.38 3.2x
0.02 (1.01 001
‘7 34 39
53 66 7x
Rearing
condttmns
‘IDGregarisation”
Fends
Significant
(P < 0.001)
Not airmticant “Calculated
Female Ax v PF. Ax v C. PF \ C Male: Au \ C. PF v C Male. Ax \’ PF
from Dirsch (lY53)
or decrease (solitarious) in chiasma frequency in the testes. However. Gillett (1975a.b) has confirmed the presence of a gregarising stimulus in the faeces of S. gregariu using other, less controversial criteria of phase change. If bacteria are indeed involved in phase transition then locusts which have been reared germ-free to adult should tend towards the solitary end of the phase spectrum. The ratios of the lengths of various parts of the body are recognised to be correlated with phase. One such ratio, F/C, which is greater in solitarious than gregarious insects, is held to be a useful practical index of phase (Dirsch, 1953). Therefore we have determined this ratio for males and females of each of the three treatments (Table 6). The ‘I0 gregarisation was also calculated with reference to published values of F/C obtained from extreme solitary and gregarious locusts. which are found only in the field (Dirsch. 1953). The F/C ratio of axenic females was significantly greater than that of conventional and parasite-free females. Similarly the F/C ratio of axenic males was significantly greater than that of conventional males. though the difference between axenic and parasitefree males was not significant. Finally both conventional males and females had significantly smaller F/C ratios than their parasite-free counterparts. Therefore. based on F/C index, the three treatments differed significantly in phase status with the degree of gregarisation in the order: females. conventional > parasite-free > axenic: males. conventional > parasite-free = axenic. Contaminution
of’ the isolutor
On four occasions the isolator became contaminated with microorganisms that appeared to cause epizootics so that the cultures were lost by the 4th instar. The organisms, isolated as monocultures in each case, were: Pseudomonas aeruginosu. a Penicillium sp. and Bacillus suhtilis (on two occasions). DISCUSSION
The gut bacterial flora does not appear to contribute significantly to laboratory desert locust nutrition, as axenic insects appeared to develop and reproduce normally. Not only did similar proportions of parasite-free and axenic insects become adult.
but mature adults of the two treatments were of similar weight and had similar sized fat bodies; with one exception, the axenic males were significantly heavier than parasite-free males. An indication perhaps that the bacterial flora may even have a slightly debilitating effect on its host. Conventional locusts were markedly inferior to the other treatments with respect to all three parameters. These insects were infected with two protozoan parasites, Malarnebu locustae and Gregarina garnhami. Gregarine parasites are believed to be relatively harmless to their hosts (Canning. 1956; Henry, 1968) thus the suboptimal performance of conventional insects is presumably due to the effects of Malameba. Axenic insects exhibited a different pattern of mortality during development to that observed in parasite-free controls, with a greater proportion of deaths occurring in later rather than in earlier instars. The reason for this is not known. Perhaps under normal conditions physiologically weak or defective insects succumb to an infection within the first few weeks of life. In an environment devoid of microbial life such individuals may continue to develop until physiological malfunction causes death later in larval life. The axenic populations of locusts lost developmental synchrony, so that the last ecdysis was spread over 6 rather than 3 days as in the parasitefree animals. Conventional locusts also exhibited a marked desynchronisation of development which may be due to the effects of the parasite, M. locustae, certainly Tobe and Pratt (1975) found that synchronous, rapid reproductive maturation in populations of Schistocrrca gregaria was only possible in the absence of Malumeha. However, the axenic animals were free from parasites. Desynchronisation has also previously been noted when gregarious locusts are transferred to solitarising conditions (Gillett and Phillips, 1977). Adult axenic locusts showed further signs of solitarisation. Based on the F/C ratio female locusts of three treatments differed significantly in phase status, with the degree of gregarisation in the order, conventional > parasite-free > axenic. That the axenic locusts were solitarised compared to the parasite-free controls, which had bacteria in their guts, is in accord with Nolte’s hypothesis that gut bacteria are responsible for supplying a precursor of locustol (Nolte, 1977). However, the parasitised
Germ-free locusts locusts were also more gregarious than their parasitefree counterparts. Since the parasitised locusts were reared in the same constant temperature room as the stock culture they may have been exposed to a greater concentration of locust01 than the parasite-free locusts which were produced in relative isolation. The situation is further complicated because it is apparent that locusts with Malameba also have more bacteria; axenic locusts have no bacteria, parasite-free have many (2.2 x 106/ml in the hindgut) but significantly fewer than Malameba infected locusts (2.1 x lO*/ml in the hindgut) (Table 5). If gut bacteria produce a pheromone that has a gregarising effect, a correlation between the degree of gregarisation and the size of the bacterial flora would be expected. Parasitised locusts would be expected to be more gregarious than parasite-free insects, as observed. Clearly the phase status of locusts infected with the parasite but without gut bacteria ought to be determined, in order to discount an effect of Malameba on phase. However, several attempts to infect axenic locusts with surface sterilised cysts have proved unsuccessful (though we have had no problems infecting parasite-free locusts [Hunt and Charnley, unpubl.]). It is tempting to speculate that this is because bacteria may directly or indirectly cause the excystment of Mulameba as it is not known what controls this event (Donaldson, 1971). In conclusion it has been shown in the present study that a lack of bacteria in crowded populations of locusts resulted in a significant increase in F/C ratio amongst females and a loss of developmental synchrony in the population as a whole. Both of these phenomena are characteristic of solitarisation and are consistent with Nolte’s hypothesis that bacteria are involved in desert locust phase transition. However, locust phases are characterised essentially by the behavioural tendencies of the insects (Uvarov, 1966) and therefore firm conclusions about the phase status of axenic locusts must await assessments of social aggregation (Gillett, 1975a). The levels of protein, lipid and carbohydrate in the blood of axenic and parasite-free insects were not significantly different. However, the lipid and carbohydrate concentrations of conventional locusts did not exhibit a consistent relationship with those of the other two treatments, in that the conventional lipid titre was significantly lower than the axenic but was not significantly different from the parasite-free, whereas the conventional carbohydrate titre was significantly higher than the parasite-free but not significantly different from the axenic. The problem may lie in the absence of a treatment consisting of parasitised but bacteria-free locusts, without which it is not possible to determine any co-operative effects of parasites and bacteria on blood lipid and carbohydrate. Unfortunately, as stated earlier, we have not been able to infect axenic locusts with cysts of M. locustae. The atrophy of the fatbody in conventional locusts is presumably due to the effects of M. locustae and confirms the observations of Papillon and Cassier (1978). No difference was found between conventional and axenic treatments with midgut carbohydrase activity. This is not consistent with Strebler’s (1978) hypothe-
483
sis that the activities of the gut flora restrict enzyme titres. However, his conclusions are based solely on the effects of starvation and antibiotic treatment he did not support his hypothesis with any viable counts of gut bacteria. In contrast to the midgut, ix-glucosidase activity of the hindgut was directly related to the density of bacteria, but in an inverse manner to that expected on the basis of Strebler’s hypothesis. The virtual absence of cc-glucosidase from axenic hindguts suggests that activity in this location is largely of bacterial origin. Since the hindgut cuticle of the desert locust is poorly permeable to molecules the size of glucose (Maddrell and Gardiner, 1980) it seems unlikely that the products of hindgut digestion contribute to host metabolism. In any case hindgut cr-glucosidase is only a small proportion of the total gut activity (Evans and Payne, 1964), and is unlikely to be significant. Indeed it is not a prerequisite of normal growth, as axenic insects develop without it. On four occasions the isolator became contaminated with microorganisms which resulted in the death of the culture in each case. One of the organisms responsible was the bacterium Pseudomonas aeruginosa; though a known insect pathogen, it often occurs harmlessly in insect guts (Bucher and Stephens, 1957). A Penicillium sp. and the bacterium Bacillus subtilis also appeared to cause epizootics. Neither of these organisms is recognised as an insect pathogen. though some species of Penicillium have been known to invade opportunistically through wounds (Vago, 1963). These results suggest that axenic locusts are more susceptible to infection than conventional locusts. There are two possible reasons for this. Either axenic locusts are deprived of a non-specific immunisation normally carried out by the resident gut flora. or the gut microflora themselves subserve a protective function by out-competing potentially harmful organisms. The former possibility seems unlikely because axenic and conventional locusts are equally susceptible to a topical dose of conidia of the parasitic fungus Metarhizium anisopliae (Dillon and Charnley, unpubl.). Other authors have observed enhanced susceptibility of axenic insects to diseasecausing organisms and have attributed this to the absence of competition from the resident gut microbes (Goodwin, 1968). Such a phenomenon is well documented for vertebrate gut flora interactions (Tannock, 1984) and it would seem to be the most appropriate explanation for the present results. Indeed we have recently shown that the bacterial flora of Schistocerca interferes with the invasiveness of an inoculum of M. anisopliae in the locust gut by producing an antimicrobial toxin (Dillon and Charnley, 1983). The technique for rearing locusts described here was in routine use in our laboratory for 3 years. However, subsequently a number of axenic populations failed to develop normally. This appears to have been due to an ageing consignment of vacuum packed irradiated bran (> 6 months old) which was nutritionally suboptimal. In order to ensure that this problem does not recur we now incorporate a double vitamin supplement (Table (1) into the bran prior to irradiation. Using this modified diet we have had no further problems.
A. K. CHARNLEY et al
484
Acknowledgements-J. H. and R. J. D. acknowledge the support of SERC studentships. We would like to thank Mr M. A. Thompson and Mr N. Weston for technical assistance, DRG packaging (Bristol) for vacuum packing the diet and Drs R. Fuller and D. J. Jayne-Williams for much advice and gifts of peracetic acid. REFERENCES
Aizawa K. (1939) Carbohydrases II Enzymic hydrolysis of p-nitrophenol /I-galactoside. Enqmologiu 6, 321-324. Bucher G. E. and Stephens J. M. (1957) A disease of grasshoppers caused by the bacterium Pseudomonas aeruginosa (Schroter) Migula. Can. J. Microbial. 3, 61 l-625. Canning E. U. (1956) A new eugregarine of locusts Gregarina garnhami N. sp. parasitic in Schistocerca gregaria Forsk. J. Protozool. 43, X%62. Cavanagh G. C. (1963) The use of the Dadd synthetic diet as a food for adult Schistocerca gregaria (Forsk) and the effects of some additions and modifications to it. J. Insect Physiol. 9, 759-775. Coates M. E.. Ford J. E., Gregory M. E. and Thompson S. Y. (1969) Effects of Gamma-irradiation on the vitamin content of diets for laboratory animals Lab. Anim. 3. 39-49. Coates M. E. and Fuller R. (1977) The gnotobiotic animal in the study of gut microbiology. In Microbial Ecolog) of the Cur (Ed. by Clarke R. T. J. and Bauchop T.). pp. 31 l-346. Academic Press. Dadd R. H. (1963) Feeding behaviour and nutrition in grasshoppers and locusts. Adr. Insect Physiol. I. 477111. Dawson R. M. C.. Elliot D. C.. Elliot W. H. and Jones K. M. (Eds) (1969) Dara .f‘or Biochemical Research. Clarendon Press. Dearn J. M. (1974a) Phase transformation and chiasma frequency variation in locusts, 1. Schistocerca gregaria. Chromosoma 45, 321-338. Dearn J. M. (1974b) Phase transformation and chiasma frequency variation in locusts II. Locusfa migratoria. Chromosoma 45, 339-352. Dillon R. J. and Charnley A. K. (1983) Per OSinfection of axenic desert locusts with the fungus Metarhizium anisopliae. 3rd Inr. S.vmp. micr. Ecol., Absi.. 27 pp. Dirsch V. M. (1953) Morphometrical studies on phases of the desert locust Schistocerca gregaria (Forskal). AnfiLOCUSIBull. (Lond.) 16. Donaldson J. M. I. (1971) Attempts to hatch cysts of Malameba locustae (King and Taylor) or control them with drugs. Phyfophvlactica 3, 1033106. Evans W. A. L. and Payne D. W. (I 964) Carbohydrases of the alimentary tract of the desert locust Schistocerca gregaria Forsk. J. Insect Physiol. 10, 6577674. Fuller R. (1968) The routine microbiological control of germ-free isolators In The Germ-Free Animal in Research (Ed. by Coates M. E.), pp. 37-45. Academic Press. Gillett S. D. (1975a) Changes in the social behaviour of the desert locust Schistocerca gregaria in response to the gregarizing pheromone. Anim. Behar. 23, 494503. Gillett S. D. (1975b) The action of the gregarisation pheromone on 5 non-behavioural characters of phase polymorphism of the desert locust, Schistocerca gregaria (Forskal). Acrida 4, 137-150. Gillett S. D. and Phillips M. L. (1977) Faeces as a source of a locust gregarisation stimulus: Effects on social aggregation and on cuticular colour of nymphs of the desert locust Schisrocerca gregaria. Acrida 6, 279-286. Goldsworthv G. J. (1969) Hyperglycaemic factors from the corpus cardiacurn of Locu.%z m&atoria. J. Insect Ph,vsiol. 15, 2131-2140. Goodwin R. H. (1968) Nonsporeforming bacterta in the armyworm Pseudoletia unipunctata. under gnotobiotic conditions. J. Inrerr. Path. 11. 358-370.
Gordon R. and Webster J. M. (1971) Mermis nigrescens: Physiological relationship with its host, the adult desert locust, Schistocerca gregaria. Expl Parasir. 29, 66-19. Hamilton A. G. (1955) Parthenogenesis in the desert locust (Schisfocerca gregaria Forsk.) and its possible effect on the maintenance of the species. Proc. R. em. Sot. Lond. A. 30. 103-114. Henry J. E. (1968) Malameba locusme and its antibiotic control in grasshopper cultures. J. Inrert. Path. 11, 224233.
Hill L. and Goldsworthy Cl. J. (1970) The utilization of reserves during starvation of larvae of migratory locust. Camp. Biochem. Phvsiol. 36, 61-70. House H. L. (1972) Insect nutrition. In Infernafional Encyclopedia of Food and Nutrition (Ed. by Fiennes R. N.). pp. 513-573. Pergamon Press. Hunt J. and Charnley A. K. (1981) Abundance and distribution of the gut flora of the desert locust, Schisfocerca gregaria. J. Inrert. Path. 38, 3788385. Hunter-Jones P. (1966) Rearing and Breeding Locusts in the Laborarory. Centre for Overseas Pest Research Publn. London. Itzhaki R. F. and Gill D. M. (1964) A micro-biuret method for estimating proteins. Analyt. Biorhem. 9, 401-410. Jayne-Williams D. J. and Hewitt D. (1972) The relationship between the intestinal microflora and the effects of diets containing raw navy beans (Phaseolus uulgaris) on the growth of Japanese quail (Coturnh coturnix japonica). J. appl. Barr. 35, 331-344. Ley F. J.. Bleby J., Coates M. E. and Paterson J. S. (1969) Sterilization of laboratory animal diets using gamma radiation. Lab. .4nim. 3, 221-254. Louveaux A., Mainguet A. M. and Gillon Y. (1980) Feeding locusts on freeze-dried plants; a new rearing method for herbivorous insects. Enromologia esp. appl. 27, 2555259. Maddrell S. H. P. and Gardiner B. 0. C. (1980) The permeability of the cuticular lining of the insect alimentary canal. J. esp. Biol. 85, 227-237. Maddrell S. H. P. and Klusuwan S. (1973) Fluid secretion by preparations of Malpighian tubules of the desert locust Schi.rtocrrcu areaaria. J. Insecr Phvsiol. 19. 1369-l 376. ., Marais J. P.. De Wit J. L. and Quicke’G. V. (1966) A critical examination of the Nelson-Somogyi method for the determination of reducing sugars. Anaiyf. Biochem. 15, 3733381. Nelson N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. biol. C/rem. 153, 375-380. Nolte D. J. (1963) A pheromone for melanization of locusts. Nature 200, 660-661. Nolte D. J. (1973) The relationship of locust species to locustol. Acrida 2, 53-60. Nolte D. J. (1977) The action of locustol. J. Insect Ph.vsiol. 23, 899-903. Nolte D. J.. Eggers S. J. and May I. R. (1973) A locust pheromone: locustol. J. Insect Physiol. 19. 1547-l 554. Nolte D. J.. May I. R. and Thomas B. M. (1970) The gregarisation pheromone of locusts. Chromosoma 29, 462-473. Ollett W. S. (1947) A method for staining both Grampositive and Gram-negative bacteria in sections J. Pathol. Bacterial. 59, 357-358. Papillon M. and Cassier P. (1978) Perturbations morphologiques et physiologiques dues a la presence du protozaine parasite Malameba locusrae (King and Taylor) chez Schisrocerca gregaria (Forsk.) Acrida 7, 101-l 14. Roe J. H. (1955) The determination of sugar in blood and spinal fluid with anthrone reagent. J. biol. Chem. 212, 3355343. Sokal R. R. and Rohlf F. J. (1969) Bionretr_v. Principles and Practices of Statistics in Biological Research. Freeman. London. Strebler G. ( 1978) Incidence du jeune et de la presence de
Germ-free microorganisme dans le tube digestif sur l’activite osidasique disgestive de Schisrocerca gregaria Forsk. Acrida 8, 35-46. Tannock G. W. (1984) Control of gastrointestinal pathogens by normal flora. In Current Perspectives in Microbial Ecology (Ed. by Klug M. J. and Reddy C. A.), pp. 37443830. American Society for Microbiology. Tobe S. S. and Pratt G. E. (1975) Corpus allatum activity in LTitroduring ovarian maturation in the desert locust Schisiocerca gregaria. J. exp. Biol. 62, 6i t-627.
locusts
485
Uvarov B. (1966) Grasshoppers and Locusts. Vol. I. Cambridge University Press. Vago C. (1983) Predispositions and interactions in insect diseases. In Insect Pathology (Ed. by Steinhaus E. A.), Vol. I, pp. 339-379. Academic Press. Ziillner N. and Kirsch K. (1962) Uber die quantitative bestimmung von lipoiden (mikromethode) mittels de vielen natulichen lipoiden (allen bakannten plasmalipoiden) gemeinsamen sulfophosphovanilhn-reaktion. Z. ges. Exp. Med. 135, 545-561.
Physiol. Vol. 31. No. 6. pp. 477485, 1985 Printed in Great Britain. All rights reserved
J. Insect
THE GERM-FREE
CULTURE
SCHISTOCERCA A. K. School of Biological
CHARNLEY,
Sciences,
University
OF DESERT
LOCUSTS,
GREGARIA
J. HUNT
of Bath,
and R. J. Claverton
DILLON
Down,
(Received 16 August 1984: revised 7 November
Bath BA2 7AY, U.K.
1984)
Abstract-A technique is described for rearing germ-free desert locusts. Axenic insects appeared to develop and reproduce normally, therefore it is concluded that the gut bacterial flora does not contribute significantly to locust nutrition. However, it is suggested that the gut flora may benefit its host by contributing to the locust’s defences against attack by pathogenic microorganisms. The results of morphometric measurements are discussed in terms of the bacterial origin of the so called phase pheromone. locustol. No evidence was found to support the hypothesis that microbial activity restricts gut carbohydrase. Key Word Index: Locust,
Schistocerca. bacteria,
axenic
MATERIALS
INTRODUCTION
In a previous publication we reported an abundant bacterial flora in the gut of Schistocerca gregariu (Hunt and Charnley, 1981). In particular a large population of Enterobacteriaceae was found with numbers increasing posteriorly from foregut to rectal sac. Whereas bacteria in the anterior regions of the gut were restricted to the lumen and the inside of the peritrophic membrane, bacteria in the hindgut were intimately associated with the cuticular lining particularly at the bottom of the folds in the ileum and between adjacent rectal pads. Little is known of the relationship between the desert locust and its gut bacterial flora. Bacteria have been implicated in the production of the so called gregarization pheromone, locustol. in a related species, Locusta migratoria (Nolte, 1977), and Strebler (1978) has suggested that microbial waste toxins may limit carbohydrase activity in the gut of S. gregaria. However, the evidence for these observations is largely circumstantial. The object of the present work was to devise a technique for culturing S. gregaria axenically and to effect a preliminary physiological characterization of germ-free insects with particular regard to the work of Nolte (1977) and Strebler (1978). The protozoan parasites Mulameba locustae and Gregarina garnhami are widespread if not universal problems in laboratory cultures (Canning, 1956; Henry, 1968; Tobe and Pratt, 1975). Therefore comparisons have been made between germ-free, conventional and parasite-free locusts. Many species of insect have been reared axenically on synthetic diets in conjunction with nutritional studies (House, 1972). However, the taxonomic distribution of the species reared axenically is biased towards the orders of Coleoptera, Diptera and Lepidoptera; there appear to have been few attempts to culture Orthoptera in this way. In addition, few species of insect have been reared on prospectively near-normal natural food with axenicity the matter of central interest rather than nutrition. 477
AND METHODS
Colonies of desert locusts, Schistocerca gregaria and migratory locusts, Locusta migratoria, were maintained according to the protocol of HunterJones (1966). Axenic desert locusts were reared in flexible plastic (PVC) isolators (see Fig. l), a method that has been employed extensively for producing germ-free vertebrates (see Coates and Fuller, 1977). Axenic insects (free from any detectable form of microbial life, no attempt was made to extract viruses) were compared with two groups of controls: conventional insects (taken from the stock colony) that had a “normal” bacterial flora and were infected with M. locustae and G. garnhami, and parasite-free insects that had only a bacterial flora. The last named were produced by raising axenic hoppers (see below) in isolators without bacterial filters in rooms which had not previously housed locusts. Populations of the three treatments were started off with 100 lst-instar larvae. Experiments were performed on 1Cday old adults and unless otherwise stated equal numbers of males and females were used. Sterilization
of equipment
Equipment was sterilized initially by irradiation, steam (121°C) or chemically using 2% peracetic acid (Laporte Inds.) as appropriate. During the construction of the isolator, the pvc unit and all associated items and furnishings were thoroughly sprayed with peracetic acid. A period of 2 days then elapsed before the axenic 1st-instar larvae were introduced, to allow the isolator to dry out, degradation of the sterilant and the removal of the volatile breakdown product acetic acid. Production
qf axenic
lst-instar
larvae
Seven to eight day old locust eggs were removed from egg tubes and carefully washed with distilled water to remove sand particles. Misshapen or discoloured eggs were discarded as potential reservoirs of bacteria. Batches of 4&50 eggs were surface sterilised by placing in 2”/, peracetic acid for 20 min. washed in
478
A. K. CHARNLEY PI (11.
sterile distilled water and then transferred aseptically to individual sterile tubes (50 x 10 mm) containing nutrient agar slopes (Oxoid). Eggs were incubated at 32 C to allow completion of development. The agar slopes were examined daily with the aid of a microscope so that contaminated eggs could be detected and discarded. Upon hatching the hoppers were retained in the tubes for a further 2 days during which time they ate the agar and voided faecal pellets. If the agar was still apparently free from microbial growth then the insect was regarded as axenic. The tubes containing the hoppers were then placed in the entry port (see Fig. 1) and sprayed with 2Y; peracetic acid, after 60min they were transferred into the isolator and released into the cage. Maintenunce of experimental insects All three treatments were maintained at a temperature of 32 f 3.C, under a 12 h light, 12 h dark photoperiodic regime. Control of cage humidity was not possible because of the destabilizing effect of daily additions of moist grass. In practice restricting feeding to early morning and late afternoon produced a diurnal relative humidity cycle that was constant between treatments (range 50-75:,;). All experimental groups were fed the same diet (unless otherwise stated); equal proportions of autoclaved ( 121°C for 30min) and vacuum packed y-irradiated (5 mega rads (IPI, Swindon)) freeze-dried grass and bran. Dry grass was rewetted with sterile water prior to feeding. Breeding usenic insects Aluminium tubes containing damp sand (HunterJones, 1966) were placed under the holes in the false floor of the insect cage. After l-2 egg pods had been laid the tube was capped, removed from the cage and kept in the isolator until the eggs were ready to hatch. At this time the tube was transferred to a 2.5 1 jar
within the isolator, where the hoppers were kept until the 3rd instar when they were transferred to the cage. Microbiological control oj’germ+ee
isolators
Clearly a reliable system of sterility testing is crucial for the validity of the operation. Procedures have been described by many authors (e.g. JayneWilliams and Hewitt, 1972; Benschoter and Wren, 1972). The protocol adopted in the present work was based on the recommendations of Fuller (1968). The germ-free status of insects in the isolators was confirmed after each ecdysis by placing samples of faecal pellets and damp swabs of cage and isolator into tubes of nutrient broth stocked inside the isolator. Tubes were examined daily (for 4 days) for signs of bacterial and fungal growth. Two days before the insects were removed for experimentation more extensive tests were performed. One animal was killed. its gut removed and the contents placed in a tube of nutrient broth; the rest of the cadaver was placed in a second tube of broth and both tubes were examined daily as before. Samples of faecal pellets were added to tubes of nutrient and thioglycollate broth (Oxoid, CM23). The nutrient broth tubes were incubated aerobically and the thioglycollate tubes anaerobically (Gas Generating Kit, Oxoid) at 32OC for 7 days. A sample from each tube was streaked onto nutrient and blood agars (Hunt and Charnley, 1981) and these were also incubated at 32°C for 7 days. Furthermore, the peritrophic membranes were removed from 20 faecal pellets, Gram stained (Ollett, 1947) and examined microscopically. Animals used for experiments were routinely examined for the presence of parasites including Malameba locustae and Gregurinu garnhami. Insects were considered to be Malameba-free if Malpighian tubules, gut and faecal pellets of mature adults were devoid of cysts when viewed in squash preparations and during light and scanning electron microscopy (Hunt and Charnley, 1981). No attempt was made to isolate any viruses present in the animals. If no microorganisms or parasites were detected by any of the tests the animals were considered to be axenic. Isolutiorl. enumeration und identt;fication of’ bucteriu The techniques employed described (Hunt and Charnley.
were as 1981).
previously
Lipid. curbolydrate and protein meusurements Blood was collected in 5 ~1 Drummond microcaps from a small puncture in the arthrodial membrane at the base of a hind leg. Total lipid concentration in samples was quantified using the phosphovanillin method as described by Ziillner and Kirsch (1962). Analysis of carbohydrate was performed with the anthrone method of Roe (1955). Protein was estimated by the micro-biuret method of Itzhaki and Gill ( 1964). Fig. I. Flexible pvc isolator system used to rear germ-free desert locusts. Filter-sterilized air was continually pumped through the isolator to maintain a positive pressure, thus reducing the possibility of contamination due to inward leakage of non-sterile air. Manipulations within the isolator were carried out using the gloved-sleeves. A, entry port: B, plastic isolator (position of the gloved-sleeves depicted by dashed circles): C. locust cage; D. outlet filter; E. inlet filter; G. pump; H. heater.
Assu~s fiw digestive enzyme acthit? Guts were dissected out in ice-cold saline (Maddrell and Klunsuwan, 1973). As required, midgut (tissue or contents of the lumen) and hindgut (tissue and contents of the lumen) were homogenised in distilled water, then centrifuged for 15 min at 2000 rpm. Supernatants were divided into 0.5 ml ali-
Germ-free locusts Table I. Vitamin supplement used in the development of the axenic diet 25 rg/g 25 I(& 25 rg/g 50 pg/g 100 fig/g 25 pg/g 125 pg/g 1 pg/g 250 @g/g 100 p&g 1.4 mg/g
Thiamine Folk acid Riboflavin Pantothenic acid Nicotinic acid Pyridoxine Choline chloride Biotin Inositol Ascorbic acid Carotene
“Single” vitamin supplement. quantities expressed per g of diet (from Cavanagh, 1963).
quots and kept deep frozen at -2O’C until required. The activities of tl- and fl-glucosidase were determined by the estimation of p nitrophenol liberated from the corresponding p nitrophenyl glycoside. Reaction mixtures of 2 ml 0.1 M McIlvane’s buffer pH 5.4 (Dawson et al., 1969) 0.5 ml of the substrate and 0.5 ml of the homogenate were incubated at 28°C for 15 min. The reaction was terminated by the addition of 3 ml of NH,OH (Aizawa, 1939) and the absorbance read at 405 nm. The activity of p-l,3 glucanase was determined by estimating the reducing sugar (glucose) released from laminarin using Nelson’s (1944) modification of the Somogyi method, observing the precautions outlined by Marais et al. (1966). Reaction mixtures of 2 ml of McIlvane’s buffer pH 5.3. 2 ml of 0.01% laminarin solution and 0.1 ml of homogenate were incubated for 1 h at 28°C. The reaction was stopped by adding 2ml of copper reagent (Nelson, 1944) to 2 ml of the incubate. The mixture was heated in capped tubes for 25 min at 100°C then cooled to 21°C. Two ml of arsenomolybdate reagent (Nelson, 1944) was added to the tube and mixed on a Vortex mixer until effervescence ceased. After 2 h colour development was complete and stable (Marais et al., 1966) and absorbance of appropriately diluted samples was read at 660nm. Morphometric measurements The length of a hind femur (F) and greatest width of the head (C), were measured to the nearest 0.01 mm using vernier calipers and a micrometer screw gauge respectively. The F/C ratio was then calculated (Dirsch, 1953). Statistics Statistical comparisons of data were performed using Student’s t-test (Sokal and Rohlf, 1969).
RESULTS
Microbiological status qf the guts of axenic, parasitefree and conventional locusts Locusts reared axenically were found to be free from microbial contamination when faecal pellets, swabs, excised guts and cadavers were assessed by the methods described in the previous section. Further-
479
more their guts and Malpighian tubules were free from the parasites M. locustae and G. garnhami. Animals reared in the absence of parasites had a mean of 2.2 x lo6 bacteria (Enterobacteriaceae)/ml (n = 11) in the hindgut. All conventional insects were infected with M. locustae and G. garnhami and had a hindgut bacterial density of 2.1 x 10” bacteria (Enterobacteriaceae)/ml (n = 56). Development of the axenic diet Dadd has defined the majority of the nutritional requirements for the growth and development of S. gregariu (Dadd, 1963). However, neither he nor Cavanagh (1963) were able to produce a complete synthetic diet; a small daily supplement of fresh grass was necessary to eliminate low fertility of eggs. In the light of this it was decided to use the normal laboratory diet of grass and bran as the basis of the axenic diet. Preliminary experiments showed that vacuumpacked freeze dried bran and grass (rehydrated before feeding) was efficacious, confirming the observations of Louveaux et al. (1980). Vacuum-packed freeze-dried grass and bran was sterilized by y-irradiation (5 mega rads) and batches of 100 newly hatched conventional L. migratoria and S. gregaria larvae raised on the diet to adult (diet 2) Only 2% of Locusta and 9% Schistocerca developed to adult, compared with 29 and 26% respectively of the controls reared on untreated grass and bran (diet 1). The failure of the experimental diet would seem to be due primarily to a lack of vitamins, as a vitamin supplement (Table 1) vastly increased the proportion of Locusta surviving to adult (22%, diet 3). Previous work has shown that B vitamins in particular are susceptible to irradiation (Ley et al., 1969) although the exclusion of oxygen and water, as practised here, provides some protection (Coates et al., 1969). A second experiment was performed with Schistocerca in which irradiated food was supplemented with an equal quantity of autoclaved grass (diet 6). This proved to be a better diet (10% ecdysed to adult) than the unsupplemented irradiated diet (5”,/, ecdysed to adult, diet 2). However, once again the addition of vitamins to the bran either before (14”/, ecdysed to adult, diet 5) or after (16% ecdysed to adult, diet 4) irradiation proved optimal. In both cases 1Cday old adult females were heavier than those reared on diets 1 and 2 and a greater proportion of the insects ecdysed to adult. Double quantities of vitamins were used when the supplement was added to the bran prior to sterilization, in an attempt to counter the deleterious effects of y-irradiation. This seems to have been effective because there was little difference between the efficacies of diet 4 (single quantities of vitamins added to the bran after irradiation) and diet 5 (double quantities of vitamins added to the bran before irradiation). Finally insects were reared on a mixture of irradiated bran and grass + autoclaved bran and grass (diet 7). This diet proved to be at least as nutritious as those supplemented with vitamins, in addition similar proportions of desert locust populations reared on diet 7 and on the normal diet ecdysed to adult (25 and 26% respectively), consequently diet 7
A. K.
CHARNLEY
adult population that was similar to parasite-free and conventional insects (Fig. 2). Populations of axenic locusts took twice as long as groups of parasite-free locusts to complete the metamorphic ecdysis (Fig. 2). An even more marked desynchronisation of development occurred amongst conventional insects. The mean wet weight of 1Cday old female axenic adult locusts was not significantly different from that of comparable parasite-free animals, though male axenics were in fact significantly heavier than male parasite-frees (Table 2). Males and females of both groups were significantly heavier than their conventional counter-parts. Fat body weights of male locusts followed a similar pattern to the female whole body weights viz. although there was no significant difference between axenic and parasite-free insects, both had much more fat body than the conventional insects (Table 2).
60 2
10
20
30
40
Days
Fig. 2. The development and pattern of mortality of axenic. parasite-free and conventional locusts. The graph represents the results from a typical experiment. Horizontal dotted lines indicate the spread of the metamorphic ecdysis. Abscissa, time in days after egg hatch Ordinate, O0 of the population alive after a given ecdysis (each point represents an ecdysis). l PO Axenic; n -m Parasite-free; A-A Conventional.
was adopted as the experimental of axenic insects. Growth and detlelopment
et uI
Breeding axenic locusts On one occasion 10 axenic adults (5 females and 5 males) were kept for breeding. Over a period of I7 days from the onset of laying I2 egg pods were produced. The details are given in Table 3: they compare favourably with data for conventional locusts reared on grass and bran by Hamilton (1955). There is considerable variation in the statistics of desert locust egg production reported in the literature. In contrast to Hamilton (1955), and the present work. Cavanagh (1963) found ca. 87 eggs/pod and 26 days to production of 1st pod. However, the viability of eggs laid by the axenic locusts (>70% hatch) was within the range observed by Cavanagh (70.&98.2”,). First generation axenic hoppers were successfully reared to adult and their parents were still alive after 120 days of adult life.
diet for the rearing
of‘ axenic locusts
the course of 4 experiments. each involving 100 insects per treatment. 29% of axenic locusts successfully developed to adult, compared with 279, of parasite-free and 25”,/, of conventional insects. Since there was little difference between axenic and parasite-free treatments it may be concluded that S. gregaria does not need a bacterial flora to successfully complete larval development. However, differences were observed in the pattern of mortality of the three experimental groups during development (Fig. 2). There was a high mortality rate amongst the early instars of parasite-free and conventional animals, only 50% of the initial population reached 3rd instar. The mortality rate then slowed down so that 2&40x achieved adulthood. In contrast to the other two treatments the axenic animals had a much reduced mortality in the early instars (80% survival to the 3rd instar). However, increased mortality during the later instars resulted in a final During
Concentration haemolJwiph
qf’lipid, protein and carbohydrate
The concentration of key macromolecules in the haemolymph of locusts are known to vary with the physiological state of the animal (Goldsworthy, 1969: Hill and Goldsworthy, 1970; Gordon and Webster, 1971). In the light of this we decided to look at the titres of protein, lipid and carbohydrate in axenic locusts to see if there were any indications of metabolic disruption. The results are recorded in Table 4. There were no significant differences between axenic and parasite-free insects with any of the parameters measured. However, conventional insects
Table 2. Whole insect and fat body weights of axenic. parasite
free and conventional
Weight of insect (g) Male Rearing
conditions
Axenic (Ax) Parasite-free (PF) Conventional (C) Significant
F%%TI& N
.r
SE
N
y
SE
N
2.15 I .96
0.05 0.07 0.03
44 9 42
3.31 3.03 1.33
0.09 0.15 0.12
37 12 18
87.0 19.9 5.3
16.9 15.4 2.01
10 9 7
Ax v C. PF Y C Ax Y PF Others
P
Male
SE
Not significant ‘P = 0.05-0.02.
locusts
Wt fat body (mg)
.I
1.66
< 0.001
in the
Ax v C. PF v C
Ax v C, PF v C
Ax v PF
Ax v PF
Germ-free locusts Table 3. Comparison
of axenic and conventional
Hopper period (days) Age of adult (days) when 1st pod produced Adult life (days) Eggs/Pod Incubation period (days)
There was no significant difference between conventional and axenic insects with any of the three enzymes in either tissue or contents of the midgut. Interestingly hindgut a-glucosidase activity was significantly greater in conventional animals than in animals reared axenically or parasite-free. The activity was directly related to the concentration of bacteria (Enterobacteriaceae) in the hindgut; animals which had the most bacteria also possessed the highest a-glucosidase activity (Table 5B). The opposite to that expected on the basis of Strebler’s hypothesis.
locust production
Axenic
Conventional (Hamilton, 1955)
28
28.6 21 68.3 40 14.7
30 >l?O 42” 14
‘> 709/, of the eggs hatched.
had a significantly lower lipid concentration axenic and a significantly higher carbohydrate centration than the parasite-free insects. Protein in the haemolymph of the three treatments similar. Carhohydrase
activity
Morphometric
than contitres were
in the gut
of lipid, protein
Lipid
measurements
In a series of papers Nolte and his colleagues (Nolte, 1963; Nolte, 1977; Nolte et al., 1970; Nolte et al., 1973) claimed to show that crowded locusts produced a pheromone (locustol) which affected phase polymorphism, making solitary locusts more like the gregarious form. The pheromone was nonspecific and was produced by Schistocerca gregaria, Locusta migratoria and Locustana pardalina (Nolte, 1973). It was demonstrated that antibiotic-treated, crowded Locusta exhibited solitary characteristics (Nolte, 1977) and the hypothesis was made that locust01 was produced by bacteria in the crop and was excreted with the faeces. Unfortunately, this work has received serious criticism. In particular a rigorous study by Dearn (1974a,b) failed to confirm the validity of Nolte’s principal (but not only) criterion of phase change, viz. an increase (gregarious)
Strebler (1978) found enhanced carbohydrase activity in the guts of desert locusts which had been either starved or fed antibiotics. He suggested that under normal conditions microbial waste products and toxins limited enzyme activity. We endeavoured to seek support for Strebler’s hypothesis by assaying for tl- and b-glucosidase and p 1:3 glucanase activities in the midguts and hindguts of locusts of the three treatments (see Tables 5A and B). Considerably more activity of all 3 enzymes was found in the lumen than in the tissue of the midgut. Table 4. Concentration
481
and carbohydrate Protein
(pg/ltl)
in the blood
of S. gregaria Carbohydrate
@g/PI)
@g/PI)
R
SE
N
R
SE
N
x
SE
N
Axenic (Ax) Parasite-free (PF) Conventional (C)
11.29 10.70 8.25
I.2 1.4 0.36
20 8 30
31.4 43.3 39.5
2.5 3.2 3.1
19 8 20
20.0 18.2 23.1
I .4
IO 7 18
Significant Not significant
Ax v C (P =0.02-0.01) Ax v PF, PF v C
Rearing
conditions
activity
in the locust gut Lumen activity
Tissue activity A. Midgut r-Glucosidase” Conventional Axenic 8.Glucosidase” Conventional Axenic p-1 :3 Glucanaseb Conventional Axenic Comparisons
between
treatment
R
SE
N
R
SE
N
4.39 2.2
0.73 0.67
12 I2
35.49 29.87
7.1 3.29
12 12
0.91 1.5
0.13 0.33
12 I2
13.21 13.18
0.82 1.28
12 12
0.98 0.7
0.13 0.07
12 12
3.27 3.13
0.24 0.09
12 12
not significant. Tissue + lumen activity
B. Hindgut z-Glucosidase” Axenic (Ax) Parasite-free (PF) Conventional (C)
PF v C (P = O.OW.02) Ax v PF, Ax v C
Ax v PF, Ax v C. PF v C
Table 5. Carbohydrase
2.3 1.1
‘Bacterial
density/ml
x 10’
R
SE
N
R
SE
N
0.07 0.29 I .49
0.01 0.07 0.23
10 IO 10
0 0.22 21
0 0.02 3.0
10 IO 56
All treatments significantly different from each other, for both enzyme activity and bacterial at P < 0.001. “Activity expressed as pmol p-nitrophenol released/gut region/l5 min. bActivity expressed as mg glucose released/midgut region/hour. ‘Enterobacteriaceae (Hunt and Charnley, 1981).
-
density,
482
A. K.
CHARNLEY et cd.
Table 6. Morpbometrlc
measurements
of adult locusts
FC .Y
SE
N
Axrnic (Ax) PamsIte-free (PF) Conventional (C)
3.50 3 39 3.2x
0.02 0.02 0 07
39 31 30
50 66 Xl
Muk Awnic (Ax) Parasite-free (PF) Convrntional (0
3 43 3.38 3.2x
0.02 (1.01 001
‘7 34 39
53 66 7x
Rearing
condttmns
‘IDGregarisation”
Fends
Significant
(P < 0.001)
Not airmticant “Calculated
Female Ax v PF. Ax v C. PF \ C Male: Au \ C. PF v C Male. Ax \’ PF
from Dirsch (lY53)
or decrease (solitarious) in chiasma frequency in the testes. However. Gillett (1975a.b) has confirmed the presence of a gregarising stimulus in the faeces of S. gregariu using other, less controversial criteria of phase change. If bacteria are indeed involved in phase transition then locusts which have been reared germ-free to adult should tend towards the solitary end of the phase spectrum. The ratios of the lengths of various parts of the body are recognised to be correlated with phase. One such ratio, F/C, which is greater in solitarious than gregarious insects, is held to be a useful practical index of phase (Dirsch, 1953). Therefore we have determined this ratio for males and females of each of the three treatments (Table 6). The ‘I0 gregarisation was also calculated with reference to published values of F/C obtained from extreme solitary and gregarious locusts. which are found only in the field (Dirsch. 1953). The F/C ratio of axenic females was significantly greater than that of conventional and parasite-free females. Similarly the F/C ratio of axenic males was significantly greater than that of conventional males. though the difference between axenic and parasitefree males was not significant. Finally both conventional males and females had significantly smaller F/C ratios than their parasite-free counterparts. Therefore. based on F/C index, the three treatments differed significantly in phase status with the degree of gregarisation in the order: females. conventional > parasite-free > axenic: males. conventional > parasite-free = axenic. Contaminution
of’ the isolutor
On four occasions the isolator became contaminated with microorganisms that appeared to cause epizootics so that the cultures were lost by the 4th instar. The organisms, isolated as monocultures in each case, were: Pseudomonas aeruginosu. a Penicillium sp. and Bacillus suhtilis (on two occasions). DISCUSSION
The gut bacterial flora does not appear to contribute significantly to laboratory desert locust nutrition, as axenic insects appeared to develop and reproduce normally. Not only did similar proportions of parasite-free and axenic insects become adult.
but mature adults of the two treatments were of similar weight and had similar sized fat bodies; with one exception, the axenic males were significantly heavier than parasite-free males. An indication perhaps that the bacterial flora may even have a slightly debilitating effect on its host. Conventional locusts were markedly inferior to the other treatments with respect to all three parameters. These insects were infected with two protozoan parasites, Malarnebu locustae and Gregarina garnhami. Gregarine parasites are believed to be relatively harmless to their hosts (Canning. 1956; Henry, 1968) thus the suboptimal performance of conventional insects is presumably due to the effects of Malameba. Axenic insects exhibited a different pattern of mortality during development to that observed in parasite-free controls, with a greater proportion of deaths occurring in later rather than in earlier instars. The reason for this is not known. Perhaps under normal conditions physiologically weak or defective insects succumb to an infection within the first few weeks of life. In an environment devoid of microbial life such individuals may continue to develop until physiological malfunction causes death later in larval life. The axenic populations of locusts lost developmental synchrony, so that the last ecdysis was spread over 6 rather than 3 days as in the parasitefree animals. Conventional locusts also exhibited a marked desynchronisation of development which may be due to the effects of the parasite, M. locustae, certainly Tobe and Pratt (1975) found that synchronous, rapid reproductive maturation in populations of Schistocrrca gregaria was only possible in the absence of Malumeha. However, the axenic animals were free from parasites. Desynchronisation has also previously been noted when gregarious locusts are transferred to solitarising conditions (Gillett and Phillips, 1977). Adult axenic locusts showed further signs of solitarisation. Based on the F/C ratio female locusts of three treatments differed significantly in phase status, with the degree of gregarisation in the order, conventional > parasite-free > axenic. That the axenic locusts were solitarised compared to the parasite-free controls, which had bacteria in their guts, is in accord with Nolte’s hypothesis that gut bacteria are responsible for supplying a precursor of locustol (Nolte, 1977). However, the parasitised
Germ-free locusts locusts were also more gregarious than their parasitefree counterparts. Since the parasitised locusts were reared in the same constant temperature room as the stock culture they may have been exposed to a greater concentration of locust01 than the parasite-free locusts which were produced in relative isolation. The situation is further complicated because it is apparent that locusts with Malameba also have more bacteria; axenic locusts have no bacteria, parasite-free have many (2.2 x 106/ml in the hindgut) but significantly fewer than Malameba infected locusts (2.1 x lO*/ml in the hindgut) (Table 5). If gut bacteria produce a pheromone that has a gregarising effect, a correlation between the degree of gregarisation and the size of the bacterial flora would be expected. Parasitised locusts would be expected to be more gregarious than parasite-free insects, as observed. Clearly the phase status of locusts infected with the parasite but without gut bacteria ought to be determined, in order to discount an effect of Malameba on phase. However, several attempts to infect axenic locusts with surface sterilised cysts have proved unsuccessful (though we have had no problems infecting parasite-free locusts [Hunt and Charnley, unpubl.]). It is tempting to speculate that this is because bacteria may directly or indirectly cause the excystment of Mulameba as it is not known what controls this event (Donaldson, 1971). In conclusion it has been shown in the present study that a lack of bacteria in crowded populations of locusts resulted in a significant increase in F/C ratio amongst females and a loss of developmental synchrony in the population as a whole. Both of these phenomena are characteristic of solitarisation and are consistent with Nolte’s hypothesis that bacteria are involved in desert locust phase transition. However, locust phases are characterised essentially by the behavioural tendencies of the insects (Uvarov, 1966) and therefore firm conclusions about the phase status of axenic locusts must await assessments of social aggregation (Gillett, 1975a). The levels of protein, lipid and carbohydrate in the blood of axenic and parasite-free insects were not significantly different. However, the lipid and carbohydrate concentrations of conventional locusts did not exhibit a consistent relationship with those of the other two treatments, in that the conventional lipid titre was significantly lower than the axenic but was not significantly different from the parasite-free, whereas the conventional carbohydrate titre was significantly higher than the parasite-free but not significantly different from the axenic. The problem may lie in the absence of a treatment consisting of parasitised but bacteria-free locusts, without which it is not possible to determine any co-operative effects of parasites and bacteria on blood lipid and carbohydrate. Unfortunately, as stated earlier, we have not been able to infect axenic locusts with cysts of M. locustae. The atrophy of the fatbody in conventional locusts is presumably due to the effects of M. locustae and confirms the observations of Papillon and Cassier (1978). No difference was found between conventional and axenic treatments with midgut carbohydrase activity. This is not consistent with Strebler’s (1978) hypothe-
483
sis that the activities of the gut flora restrict enzyme titres. However, his conclusions are based solely on the effects of starvation and antibiotic treatment he did not support his hypothesis with any viable counts of gut bacteria. In contrast to the midgut, ix-glucosidase activity of the hindgut was directly related to the density of bacteria, but in an inverse manner to that expected on the basis of Strebler’s hypothesis. The virtual absence of cc-glucosidase from axenic hindguts suggests that activity in this location is largely of bacterial origin. Since the hindgut cuticle of the desert locust is poorly permeable to molecules the size of glucose (Maddrell and Gardiner, 1980) it seems unlikely that the products of hindgut digestion contribute to host metabolism. In any case hindgut cr-glucosidase is only a small proportion of the total gut activity (Evans and Payne, 1964), and is unlikely to be significant. Indeed it is not a prerequisite of normal growth, as axenic insects develop without it. On four occasions the isolator became contaminated with microorganisms which resulted in the death of the culture in each case. One of the organisms responsible was the bacterium Pseudomonas aeruginosa; though a known insect pathogen, it often occurs harmlessly in insect guts (Bucher and Stephens, 1957). A Penicillium sp. and the bacterium Bacillus subtilis also appeared to cause epizootics. Neither of these organisms is recognised as an insect pathogen. though some species of Penicillium have been known to invade opportunistically through wounds (Vago, 1963). These results suggest that axenic locusts are more susceptible to infection than conventional locusts. There are two possible reasons for this. Either axenic locusts are deprived of a non-specific immunisation normally carried out by the resident gut flora. or the gut microflora themselves subserve a protective function by out-competing potentially harmful organisms. The former possibility seems unlikely because axenic and conventional locusts are equally susceptible to a topical dose of conidia of the parasitic fungus Metarhizium anisopliae (Dillon and Charnley, unpubl.). Other authors have observed enhanced susceptibility of axenic insects to diseasecausing organisms and have attributed this to the absence of competition from the resident gut microbes (Goodwin, 1968). Such a phenomenon is well documented for vertebrate gut flora interactions (Tannock, 1984) and it would seem to be the most appropriate explanation for the present results. Indeed we have recently shown that the bacterial flora of Schistocerca interferes with the invasiveness of an inoculum of M. anisopliae in the locust gut by producing an antimicrobial toxin (Dillon and Charnley, 1983). The technique for rearing locusts described here was in routine use in our laboratory for 3 years. However, subsequently a number of axenic populations failed to develop normally. This appears to have been due to an ageing consignment of vacuum packed irradiated bran (> 6 months old) which was nutritionally suboptimal. In order to ensure that this problem does not recur we now incorporate a double vitamin supplement (Table (1) into the bran prior to irradiation. Using this modified diet we have had no further problems.
A. K. CHARNLEY et al
484
Acknowledgements-J. H. and R. J. D. acknowledge the support of SERC studentships. We would like to thank Mr M. A. Thompson and Mr N. Weston for technical assistance, DRG packaging (Bristol) for vacuum packing the diet and Drs R. Fuller and D. J. Jayne-Williams for much advice and gifts of peracetic acid. REFERENCES
Aizawa K. (1939) Carbohydrases II Enzymic hydrolysis of p-nitrophenol /I-galactoside. Enqmologiu 6, 321-324. Bucher G. E. and Stephens J. M. (1957) A disease of grasshoppers caused by the bacterium Pseudomonas aeruginosa (Schroter) Migula. Can. J. Microbial. 3, 61 l-625. Canning E. U. (1956) A new eugregarine of locusts Gregarina garnhami N. sp. parasitic in Schistocerca gregaria Forsk. J. Protozool. 43, X%62. Cavanagh G. C. (1963) The use of the Dadd synthetic diet as a food for adult Schistocerca gregaria (Forsk) and the effects of some additions and modifications to it. J. Insect Physiol. 9, 759-775. Coates M. E.. Ford J. E., Gregory M. E. and Thompson S. Y. (1969) Effects of Gamma-irradiation on the vitamin content of diets for laboratory animals Lab. Anim. 3. 39-49. Coates M. E. and Fuller R. (1977) The gnotobiotic animal in the study of gut microbiology. In Microbial Ecolog) of the Cur (Ed. by Clarke R. T. J. and Bauchop T.). pp. 31 l-346. Academic Press. Dadd R. H. (1963) Feeding behaviour and nutrition in grasshoppers and locusts. Adr. Insect Physiol. I. 477111. Dawson R. M. C.. Elliot D. C.. Elliot W. H. and Jones K. M. (Eds) (1969) Dara .f‘or Biochemical Research. Clarendon Press. Dearn J. M. (1974a) Phase transformation and chiasma frequency variation in locusts, 1. Schistocerca gregaria. Chromosoma 45, 321-338. Dearn J. M. (1974b) Phase transformation and chiasma frequency variation in locusts II. Locusfa migratoria. Chromosoma 45, 339-352. Dillon R. J. and Charnley A. K. (1983) Per OSinfection of axenic desert locusts with the fungus Metarhizium anisopliae. 3rd Inr. S.vmp. micr. Ecol., Absi.. 27 pp. Dirsch V. M. (1953) Morphometrical studies on phases of the desert locust Schistocerca gregaria (Forskal). AnfiLOCUSIBull. (Lond.) 16. Donaldson J. M. I. (1971) Attempts to hatch cysts of Malameba locustae (King and Taylor) or control them with drugs. Phyfophvlactica 3, 1033106. Evans W. A. L. and Payne D. W. (I 964) Carbohydrases of the alimentary tract of the desert locust Schistocerca gregaria Forsk. J. Insect Physiol. 10, 6577674. Fuller R. (1968) The routine microbiological control of germ-free isolators In The Germ-Free Animal in Research (Ed. by Coates M. E.), pp. 37-45. Academic Press. Gillett S. D. (1975a) Changes in the social behaviour of the desert locust Schistocerca gregaria in response to the gregarizing pheromone. Anim. Behar. 23, 494503. Gillett S. D. (1975b) The action of the gregarisation pheromone on 5 non-behavioural characters of phase polymorphism of the desert locust, Schistocerca gregaria (Forskal). Acrida 4, 137-150. Gillett S. D. and Phillips M. L. (1977) Faeces as a source of a locust gregarisation stimulus: Effects on social aggregation and on cuticular colour of nymphs of the desert locust Schisrocerca gregaria. Acrida 6, 279-286. Goldsworthv G. J. (1969) Hyperglycaemic factors from the corpus cardiacurn of Locu.%z m&atoria. J. Insect Ph,vsiol. 15, 2131-2140. Goodwin R. H. (1968) Nonsporeforming bacterta in the armyworm Pseudoletia unipunctata. under gnotobiotic conditions. J. Inrerr. Path. 11. 358-370.
Gordon R. and Webster J. M. (1971) Mermis nigrescens: Physiological relationship with its host, the adult desert locust, Schistocerca gregaria. Expl Parasir. 29, 66-19. Hamilton A. G. (1955) Parthenogenesis in the desert locust (Schisfocerca gregaria Forsk.) and its possible effect on the maintenance of the species. Proc. R. em. Sot. Lond. A. 30. 103-114. Henry J. E. (1968) Malameba locusme and its antibiotic control in grasshopper cultures. J. Inrert. Path. 11, 224233.
Hill L. and Goldsworthy Cl. J. (1970) The utilization of reserves during starvation of larvae of migratory locust. Camp. Biochem. Phvsiol. 36, 61-70. House H. L. (1972) Insect nutrition. In Infernafional Encyclopedia of Food and Nutrition (Ed. by Fiennes R. N.). pp. 513-573. Pergamon Press. Hunt J. and Charnley A. K. (1981) Abundance and distribution of the gut flora of the desert locust, Schisfocerca gregaria. J. Inrert. Path. 38, 3788385. Hunter-Jones P. (1966) Rearing and Breeding Locusts in the Laborarory. Centre for Overseas Pest Research Publn. London. Itzhaki R. F. and Gill D. M. (1964) A micro-biuret method for estimating proteins. Analyt. Biorhem. 9, 401-410. Jayne-Williams D. J. and Hewitt D. (1972) The relationship between the intestinal microflora and the effects of diets containing raw navy beans (Phaseolus uulgaris) on the growth of Japanese quail (Coturnh coturnix japonica). J. appl. Barr. 35, 331-344. Ley F. J.. Bleby J., Coates M. E. and Paterson J. S. (1969) Sterilization of laboratory animal diets using gamma radiation. Lab. .4nim. 3, 221-254. Louveaux A., Mainguet A. M. and Gillon Y. (1980) Feeding locusts on freeze-dried plants; a new rearing method for herbivorous insects. Enromologia esp. appl. 27, 2555259. Maddrell S. H. P. and Gardiner B. 0. C. (1980) The permeability of the cuticular lining of the insect alimentary canal. J. esp. Biol. 85, 227-237. Maddrell S. H. P. and Klusuwan S. (1973) Fluid secretion by preparations of Malpighian tubules of the desert locust Schi.rtocrrcu areaaria. J. Insecr Phvsiol. 19. 1369-l 376. ., Marais J. P.. De Wit J. L. and Quicke’G. V. (1966) A critical examination of the Nelson-Somogyi method for the determination of reducing sugars. Anaiyf. Biochem. 15, 3733381. Nelson N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. biol. C/rem. 153, 375-380. Nolte D. J. (1963) A pheromone for melanization of locusts. Nature 200, 660-661. Nolte D. J. (1973) The relationship of locust species to locustol. Acrida 2, 53-60. Nolte D. J. (1977) The action of locustol. J. Insect Ph.vsiol. 23, 899-903. Nolte D. J.. Eggers S. J. and May I. R. (1973) A locust pheromone: locustol. J. Insect Physiol. 19. 1547-l 554. Nolte D. J.. May I. R. and Thomas B. M. (1970) The gregarisation pheromone of locusts. Chromosoma 29, 462-473. Ollett W. S. (1947) A method for staining both Grampositive and Gram-negative bacteria in sections J. Pathol. Bacterial. 59, 357-358. Papillon M. and Cassier P. (1978) Perturbations morphologiques et physiologiques dues a la presence du protozaine parasite Malameba locusrae (King and Taylor) chez Schisrocerca gregaria (Forsk.) Acrida 7, 101-l 14. Roe J. H. (1955) The determination of sugar in blood and spinal fluid with anthrone reagent. J. biol. Chem. 212, 3355343. Sokal R. R. and Rohlf F. J. (1969) Bionretr_v. Principles and Practices of Statistics in Biological Research. Freeman. London. Strebler G. ( 1978) Incidence du jeune et de la presence de
Germ-free microorganisme dans le tube digestif sur l’activite osidasique disgestive de Schisrocerca gregaria Forsk. Acrida 8, 35-46. Tannock G. W. (1984) Control of gastrointestinal pathogens by normal flora. In Current Perspectives in Microbial Ecology (Ed. by Klug M. J. and Reddy C. A.), pp. 37443830. American Society for Microbiology. Tobe S. S. and Pratt G. E. (1975) Corpus allatum activity in LTitroduring ovarian maturation in the desert locust Schisiocerca gregaria. J. exp. Biol. 62, 6i t-627.
locusts
485
Uvarov B. (1966) Grasshoppers and Locusts. Vol. I. Cambridge University Press. Vago C. (1983) Predispositions and interactions in insect diseases. In Insect Pathology (Ed. by Steinhaus E. A.), Vol. I, pp. 339-379. Academic Press. Ziillner N. and Kirsch K. (1962) Uber die quantitative bestimmung von lipoiden (mikromethode) mittels de vielen natulichen lipoiden (allen bakannten plasmalipoiden) gemeinsamen sulfophosphovanilhn-reaktion. Z. ges. Exp. Med. 135, 545-561.