Chemoreception and food selection in locusts

35

TINS - February 1981

Chemoreception and food selection in locusts W. M. Blaney Food selection mechanisms, and in particular the role of secondary plant compounds, are being investigated in two locust species which respond differently to these compounds and to the plants in which they occur. Studies or feeding behaviour and of the fine structure and neurophysiology o f the sense organs involved are being used to examine these interspecies differences, Locusts have attracted the attention of scientists for decades, partly because of their formidable eating habits, making them a very significant agricultural pest, and partly because they are a very useful subject for the investigation of insect physiology. This study encompasses both aspects of our preoccupation with locusts. Many think of 'the locust' as a single species which eats green plants voraciously and indiscriminately. In fact there are several species and they are selective in their choice of food, some more so than others. I have worked on two species, chosen because they differ in the range of plants included in their diet. Schistocerca gregaria, the desert locust, is polyphagous, eating a wide range of grasses and many dicotyledons; Locusta migratoria, the migratory locust, is oligophagous, restricting its choice of food

to the grasses. The approach has been to quantify the insects' activities under different feeding conditions so as to identify and analyse elements of the complex behaviour patterns involved, to determine which organs are used in food selection, and finally to correlate this information with studies of the fine structure and neurophysiology of these organs. Locusts normally feed for 5-15 min periods separated by intervals of about 1 h during which feeding does not occur. The critical testing of plant material is carried out by contact chemoreceptors on the maxillary palps (Fig. 1) and inside the pre-oral cavity. In insects which have not been deprived of food for a long period, the sensilla on the palp tips may play a key role in determining whether potential food material is accepted U. The sensilla

)' f~

i

There are about 400 trichoid chemoreceptor sensilla on the flexible dome of each maxillary palp tip in fifth instar nymphs of both species (Fig. 2). Some 5 % are olfactory sensiUa of uncertain function; a further 5% are gustatory, responding to dissolved chemicals, plant

volatiles and some insect alarm pheromone constituents, but whose role in food selection is not clear. The remaining 90% are contact chemoreceptors, usually having six neurones, one of which may be a mechanoreceptor (Fig. 3). Sensilla of this type have a minute crest at the tip of the hair surrounding a single pore which is capable of opening and closing. When the sensilla are palpated on the plant surface, contact is made, through the pore, between the dendrites within the hair and chemicals from the plants. These crested sensilla, being in the majority, have been the subject of a special study. Measuring the electrical resistance across the palp tip shows that the pores close after feeding in response to a hormonal stimulus induced by gut stretch receptors and, as the interfeed period ends, the pores open again ~. The exact mechanism of pore movement is not known nor is its significance obvious. The dendrites are protected and non-functional when the pore is closed and it may be important to limit the periods of functioning of the sensory cells. The cells are not renewed, in contrast with those of vertebrates, and must last the lifetime of the insect. Palpation The functioning of the receptor cells has been studied by standard electrophysiology with an indifferent electrode in the insect's body and a recording electrode in a glass capillary containing weak electrolyte and stimulating chemicals. During recording, the capillary is manipulated to cover the open tip of a single hair. In normal palpation the sensilla make rapid (10-15 Hz) brief (20 ms) contacts with the test substrate and this was simulated by mounting

J

Fig. I. A filth instar locust (Locusta migratoria) with the maxillary and labial palps showing under the head (see arrows).

Fig. 2a. The tip o f a maxillary palp bearing numerous trichoid chemoreceptor sensilla.

Fig. 2b. Scanning electron-micrograph o f palp tip sen. silia showing the crested sensilla which are in the major-

ity.

FINS - February 1981

36

a

the recording capillary on the armature of a loudspeaker driven by square wave p u l s e s at frequencies up to 10 Hz. This mode of stimulation reduces neural adaptation but otherwise does not appear to alter the neural code contained in the sensillum output 7. Consequently in most experiments the output during the first second of continuous stimulation, more conveniently obtained, has been taken to represent the 'message' of the sensillum to the CNS 4. The neural code The nature of that 'message' is an intriguing subject for investigation. It was quickly realised s that the concept of neurones selectively responsive to salt, sugar, water etc. as sometimes found in other insects, did not apply here. In different sensilla a variable n u m b e r of neurones would respond to the same simple solution; cross adaptation experiments would show the same neurones in a given sensillum responding to say, salt solution and to salt with sugar solution, albeit with different rates of response. Such specificity as did occur was more at the level of sensilla than of neurones; even so, most sensilla could not individually report unambiguously the nature of a range of different taste modalities. The code for taste quality was found to consist of the relative a m o u n t s of activity produced simultaneously in many different sensill#. This, of course, is the across-fibre analysis proposed by Pfaffman* but applied here to the output of sensilta rather than of neurones. The across-fibre hypothesis requires that (a) receptors have unique but overlapping action spectra and (b) each substance discriminated generates a

unique total pattern of response. Achievement of these requirements is hampered in the locust by the occurrence of variability in the response of a given sensillum to a given stimulus, which is in the order of - 10%. The effect of this variability has been tested by taking two solutions which are discriminated behaviourally (e.g. (i) 0.05M NaCI and (if) 0.05M NaC1 with 0.025M fructose) and recording the responses from about twenty sensilla on the same palp of one insect, giving each sensillum four separate tests with each solution. The variance between the responses of individual sensilla to a single solution is highly significant indicating that sensilla differ from each other in their response the first tenet of the across-fibre hypothesis. The variance between the responses to the different solutions is also highly significant indicating that, on the total information available in each case, the insect could differentiate between solutions - as confirmed by behavioural experiments. Further, there is a highly significant variance due to interaction, indicating that different sensilla respond in different ways to the two stimulus solutions; that is, each solution generates a different total pattern of response - the second tenet of the across-fibre hypothesis. Thus, despite the variability, the palp-tip sensilla generate a neural output which will allow discrimination so long as the output from a population of sensilla is considered. Inter-slmeific dtnerenees Both species of locusts seem to rely on this method of sensory analysis and seem to assess nutritious, palatable substances simi-

larly. However, they do show intriguing differences in the way they handle and respond to non-nutrient chemicals which occur in plants. There are some 10,000 of these so-called ~secondary' plant compounds with a scattered distribution among higher plants, sometimes with individual compounds restricted to a single plant species. Mostly they are deterrent to insects but they can act as phagostimulants in some cases. For the oligophagous L o c u s t a , most grasses lack deterrent secondary compounds but most non-grasses contain one or more compounds deterrent for L o c u s t a and such plants are not eaten. Some compounds, deterrent for L o c u s t a are ineffective against S c h i s t o c e r c a , many are only effective at much higher concentrations and some, deterrent at high concentrations, actually stimulate feeding in S c h i s t o c e r c a at lower concentrationsL Thus it would seem that the sensory system of Schistocerca makes a more detailed assessment of the nature and the concen-

Fig. 3. A diagrammanc representation of the structure of a sensillum, with neurones (central) surrounded by formative and sheath cells (shaded). For clat'ity, only one dendrite is shown reaching the hair tip.

37

TINS - February 1981 100- Relative

/~,0 II

unpalatability %

• AZs ,t~_l

• Ni,

~

100 -] Relative unpalatability %

/

•Ni~ Ni~o

~

~ A HO •Ni,o

Az~o

50

50 •

~

J

.

'~u~

bu~

SUlA

(a) Schistocerca

Ta

ATa

• Su~

St~,o

sin

(b) Locusta

~u,0

Relative sensory input (spikes/sec.)

3'O0

r

60/0

300

Relative sensory input (spikes/sec.) 1

600

Fig. 4. Correlation between relative unpalatability and relative sensory input. See footnote for explanation o f symbols.

their response to different compounds and, as with nutrients, most appear to confuse some of the solutions tested. There is no apparent single peripheral code either for acceptance or rejection but, though one would not expect a simple correlate of behaviour with peripheral sensory input, that input must be the basic information on which behavioural decisions are based. A n attempt was made to improve the correlation by modelling the sensory information available to a given locust by using the responses from several sensilla from one insect only. Thus the relative sensory input distinguishing each solution from the next is based on the actual rate of firing for the control (one compound simpler) solution, the average magnitude of change when it occurred and the actual n u m b e r of sensilla in the three categories of change (see Table I). This ensures that the actual firing differential in any one sensillum is normalised in relation to any other. The relative sensory input so obtained may be compared with the behavioural response to the same solutions (Fig. 4). In S c h i s t o c e r c a , palatability is correlated with low firing rates and small changes in sensory input reflect large changes in * The solutions used were: Sa = 0.05M NaCI; Sut = 0.05M NaCI + 0.01M sucrose; Ta = 0.05M NaCI + palatability. This may relate to the ability 0.01M sucrose + 0.002M sodium hydrogen (+) tar- of S c h i s t o c e r c a to regulate its intake over a trate; Nit = 0.05M NaCI + 0.01M sucrose + 0.002M wide range of food plants according to their nicotine hydrogen (+) tartrate; Su/= 0.05M NaCI + palatability. The exception is azadirachtin, 0.01M sucrose + 0.007M sodium sulphate; Ho = 0.05M NaCI + 0.01M sucrose + 0.007M hordenine a potent feeding inhibitor for S c h i s t o c e r c a ; hemisulphate;S/n = 0.05M NaCI + 0.0IM sucrose + here the sensory input must contain 0.002M sinigrin;Az~= 0.05M NaCI + 0.01M sucrose specific instructions for rejection. In + 17 /~g/l azadiraehtin. Additionally, the effect of L o c u s t a , sensory input correlates well with increased sugar concentration in three of these solu- behavioural response to sugar concentrations was tested by substituting the 0.01M sucrose with 0.05M sucrose(Sul, NiB,Az=) and with 0.1M suc- tion, in the same sense as with S c h i s t o c e r c a rose (Sut*, Nit,, AZ~*). Thus each solution differsfrom but, with addition of secondary comone other by the addition or lack of only one com- pounds, decreased firing signals unpalatapound. bility. Whatever the mechanism, both

tration of secondary compounds than does that of L o c u s t a . This possibility has been investigated by behavioural and neurophysiological experiments using a range of solutions containing sucrose as a phagostimulant and various secondary compounds as deterrents to which the two species react differently. The test solutions represent a compromise between the desirability of using complex mixtures akin to those occurring in plants and the need to use a series of solutions of increasing complexity to facilitate interpretation of neurophysiological responses.* Feeding responses to fibre-glass discs, each soaked with one of the test solutions, were noted and the relative unpalatability of the solutions expressed as a percentage of insects failing to attempt to feed on them at first encounter. Taking account of the measured variability in sensillum response to the first second of stimulation, the effect on sensillum firing rate of each added compound in the series of solutions can be assessed, and 'no change', 'increase' and 'decrease' sensilla for each compound identified. Sensilla differ considerably in

insects show much reduced intake in the presence of secondary compounds from plants which they would not normally eat. When the output of sensilla in each of the three response categories is compared individually, or in various combinations, with the behavioural response, further information is gainedL Thus in L o c u s t a the best correlation is with the s u m m e d input from 'increase' firers and 'no change' firers. Surprisingly, behaviour correlates well with sensilla showing no change (as judged by the experimenter), but not at all with the 'decrease' firers, which account for 50% of all sensilla. These apparent anomolies would be explained if for any one sensillum a certain output level is required before it can contribute towards palatability assessment, thus eliminating the potentially disruptive influence of the low firing sensilla. If a similar threshold level existed at a central neuronal level, the 'increase' firers alone, indicating palatability, would not correlate well with behaviour but the generally high firing rate of the 'no change' sensilla adding their input to the CNS would lift the overall level above the barrier and allow it to be modulated by the TABLE 1. Computation of relative sensoryinput 1. av. spikes/sec,for control x no. of'no change' sensilla 2. av. spikes/sec,for increasea × no. of 'increase' sensilla 3. av. spikes/sec, for decreasea x no. of 'decrease' sensilla 1 + 2 + 3 = relative sensory input (spikes/see.) a Mean percent increase/decrease converted to spikes/sec, and added to/subtracted from control level.

38

'FINS - February 1981

'increase" firers. Such a mechanism of discrimination would not be very adequate if subtle differences in a wide range of substances had to be assessed, but the diet of Locusta is virtually restricted to grasses, and when it accepts them it usually eats a fairly complete meaP. The approach described here uses only the quantitative output from sensilla, ignoring qualitative differences, and it is somewhat surprising that any correlation at all with behaviour is seen. Interaction between stimulatory and inhibitory chemicals could occur at the receptor level and the input to the CNS be determined by that conflict, or input from different 'labelled

lines" could signal inhibition and stimulation concurrently, leaving the conflict to be resolved by the CNS. It seems that both mechanisms operate, although to different extents, in the two species studied here and useful guidelines have been established for studies both at peripheral and central neuronal levels in these locusts, largely as a result of considering behavioural responses in conjunctkm with neurophysiology.

Reading list 1 Bcrnays. E, A. and Chapman, R, F. (1978) in Coevolution of plants and animals ( Harborne. J., ed.). Academic Press, London 2 Bernays, E. A., Blaney, W. M. and Chapman, R. F.

Neuronal oscillators in Ap lysia : modutmion by serotonin and cyclic AMP Irwin B. Levitan and Jack A. Benson Many rhythmic behaviours are controlled by oscillators in the nervous system, and the modulation o f such oscillators is thus. o f considerable physiological importance. In this paper two neuronal oscillators o f very different frequency found in the sea slug, Aplysia californica are discussed. One is the high frequency rhythm in the activity o f identified neurone R 15; the other is the low frequency circadian rhythm o f activity in the eye. Both are influenced by serotonin and cyclic AMP. We present a model in which the physiological response o f neurone R15 to serotonin is mediated by intracellular cyclic AMP. subject of considerable productive research and we now know a great deal about the neuronal mechanisms underlying a variety of behaviours in this animal". Several Aplysia neurones are 'membrane' oscillators known as pacemaker cells. The most extensively studied is the identified neurone RI 5 in the abdominal ganglion 1~. Its large size and ready identifiability make it particularly favourable for electrophysiological, pharmacological, and biochemical investigation. The circadian system located in the Aplysia eye 9 is an example of interaction between cytoplasmic and membrane oscillatorsL The clock, a 'cytoplasmic' oscillator involving protein synthesis1°, manifests itself by modulating the frequency of compound action potential (CAP) bursts originating An invertebrate model at the cell membranes of the secondary The nervous system of the sea slug neurones in the eye. Although the rhythms Aplysia califi~rnica (Fig. 1), has been the of R15 and the eye are different in many

Rhythmic activity is widespread in biological systems. It is observed in whole organisms, individual organs and tissues, and even in single cells TM. Biological rhythms can have periods ranging from less than a second to a day and longer. Berridge and Rapp ~ have defined two major types of cellular oscillations, one based on rhythmic changes in the properties of the surface membrane of a cell, and the other arising from changes occurring in the cytoplasmic components within the cell. Such a distinction can be somewhat arbitrary if membrane and cytoplasmic oscillators interact within the same system. Nevertheless, the division into these two classes has been useful in analysing the oscillations in a variety of tissues, including the nervous system.

~c~EIs¢~icr/Norlh-t]oliand BiomedicalPress 1981

(1972)J. Erp. Bh/. 57. 745-753 3 Blaney. W. M.(1974)J. Exp. Biol. 60, 275-293 4 Blaney, W. M. (1975) J. Exp. Biol. 62, 555-569 5 B[aney, W. M. and Chapman, R. V (197( 0 Entomol, E.tp. Appl. 13,363-376 6 Blaney. W. M., Chapman, R. F. and Wilson. A (1973)Acrida, 2, 119-137 7 Blaney. W. M. and Ducken, A. M (1975)J t=xp. Biol. 63,701 712 8 Blaney. W. M. and Winstanley, C. (1980) inlnsect

Neurobiology and Pesticide Action (Neurotox 79). pp. 383-389, Chem. Industry, London 9 Pfaffmann. C (1041) J, Cell. Comp. Physiol. 17, 243-258

W. M. Bhmey L~ a Senior Lecturer in the Department of Zoology, Birkbeck College (University of London), Malet Street, London, WCIE 7HX, U.K.

respects, they have at least one important feature in common; both can be modified by serotonin and cyclic AMP.

Circadian clock modulation by serotonin and cyclic AMP It is likely that the system generating the bursts of CAPs recorded in the optic nerve of isolated Aplysia eyes consists of a population of electrotonically coupled secondary neurones with pacemaker properties similar to those of R15 =. The effects of serotonin on this system fall into two categories. Firstly, there is an immediate and reversible decrease in the CAP output from the eye 4, and, secondly, permanent phase shifts of the metabolic circadian oscillator occur 4.s. In general, treatment with serotonin delays the rhythm when it is applied during its rising phase and advances it during the falling phase 4. The actions of cyclic AMP analogues on the eye are similar to those of serotonin ~'~.7 (Fig. 2). During the course of the experimental pulse, the C A P frequency and number of CAPs. per burst decreases, and the amplitude of individual CAPs falls suggesting that fewer action potentials are contributing to each CAP, This initial effect on the membrane oscillator is reversed by washing in normal medium (Fig. 2). The other action of cyclic AMP analogues, on the circadian metabolic oscillator, results in steady-state phase shifts of the experimental eyes in comparison with the controls (Fig. 2). These phase shifts are not reversed by washing and can therefore be regarded as a change in the circadian clock mechanism itself and not simply a transient change in the output from the clock. Eskin 7 reports that the phase