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Abstract The dual??choices tests of behavioral test were used to study the gustatory behavioral responses to caffeine of Helicoverpa armigera larvae and H. assulta larvae. Electrophysiological responses were studied by electrophysiological tip recording. Behavioral experiments showed that caffeine was a feeding deterrent for both larvae， but it showed a stronger feeding deterring effect on the oligophagous H. assulta. The electrophysiological tip??recording results showed that both H. armigera larvae and H. assulta larvae had one caffeine??sensitive feeding deterrent neuron at the medial sensilla， and the electrophysiological pulse response of H. assulta larvae was significantly stronger than that of H. armigera larvae. Therefore， caffeine had a stronger feeding deterring effect on the oligophagous H. assulta than the polyphagous H. armigera. The difference in behavioral effect was closely related to the sensitivity of to caffeine the feeding deterrent neurons at the medial sensilla.
Key words Helicoverpa armigera； H. assulta； Caffeine； Feeding deterrent； Tip??recording
Helicoverpa armigera is a worldwide agricultural pest[1]. It is also a typical polyphagous insect. The host plants include more than 200 species in over 30 families like Solanaceae， Leguminosae， and Compositae. However， its closely related species of H. assulta is an oligophagous insect， which only feeds on a few plants such as pepper and tobacco[2]. The two species of insects can detect the soluble taste compounds in the external environment mainly through the taste receptors on maxillary palpus and epipharynx， as well as the medial and lateral sensilla on the Galea[3]. Multiple taste neurons in the sensilla on the lateral sensiilla play a crucial role in feeding behavior[4]. Feeding deterrent neuron can sense the feeding deterrent in external environment， which can inhibit or even stop the feeding behavior of insects[4-5]. Caffeine is a feeding deterrent for many insect larvae[6]， but little is known about its feed deterring effects on H. armigera and H. assulta. This study compared the behavior and electrophysiological responses of the polyphagous H. armigera and the oligophagous H. assulta to caffeine， which provided a theoretical basis for understanding the mechanism of the taste perception of different feeding insects.
Materials and Methods
Materials
Plants and insects H. armigera and H. assulta were collected from the field and cultured in the laboratory for the second generation at the temperature of （25??1）??， relative humidity of 60%-70%， and photoperiod of L16： D8. After 3 instars old， the larvae were kept in the glass worm??culturing tubes， one per tube， and artificial diet was placed in the tubes. The tubes were blocked with cotton plugs. Adults were placed in cages and fed with 10% honey water. The larvae used for behavioral experiments and electrophysiological tests were reared to 4 instars normal artificial diet. The larvae were taken out from the insect tubes to at molting stage of the 4th instar and placed in empty tubes. After the larvae reached the 5 instars， they were starved for about 8 h. Preparation of feed The artificial feed formula for H. armigera and H. assulta is shown in Table 1.
Test compound and behavioral experimental medium Caffeine was purchased from Sigma??Aldrich with a purity of >99%. The behavioral experiment used whatman?k glass fiber filter paper as the feeding medium and was purchased from Sigma??Aldrich.
Methods
Behavioral test Behavioral tests were performed using dual??choices tests with glass fiber filter paper as the feeding medium. The fiber paper was punched into discs with the diameter of 1 cm. Then， 10 ??l of 10 mM caffeine solution was evenly applied to the glass fiber filter discs with a pipette， which were the treated discs， and 10 ??l of distilled water was evenly applied to other glass fiber filter discs， served as the control discs. The starved 5??instar larvae were placed in the middle of the petri dish and the tests were carried out in a greenhouse at 25 ??. Then， 2 treated filter discs and 2 control filter discs were placed on the inner edge of the petri dish sequentially[7]， and when about 50% of any of the treated discs or control discs were fed， the area of the remaining discs was scanned with an HP scanner and the surface area was calculated using Scion Image software. And the Feeding Deterrent Index （FDI） was calculated. FDI indicated that the intensity of the feeding deterrent deterring the feeding， and it was calculated using the following formula：
FDI=100 （CT）/（C+T）
Where， C indicates that feeding area of the control disc； T is the feeding area of the treated disc. The value of FDI was between 1-100， and the larger value suggested a stronger feeding deterring effect[8].
Electrophysiological test The neuro??pulse response of caffeine in the lateral and medial sensilla was tested using the electrophysiological Tip??recording method[9]. The larvae of H. armigera and H. assulta at the beginning of 5 instars were taken， and then their heads were cut quickly with a scalpel blade. Then， the head was put at one end of the control electrode， which was slightly tilt， and the lower jaw was strutted to outspread the lateral and medial sensilla. The other end of the control electrode was connected to a preamplifier （Syntech Taste Probe DTP??1， Hilversum， the Netherlands）， which was connected to a signal processing instrument （A/D??interface， Syntech IDAC??4， Hilversum， the Netherlands） together. Caffeine was dissolved in 2 mM KCl solution， which was the control solution of the electrophysiological test， and the caffeine solution of 0.1， 1 and 10 mM was made separately. The caffeine solution was then injected into the glass micropipette with a tip diameter of about 30 ??m， which was the test electrode. During the test， the nerve impulse signals of the taste neuron reaction were amplified by a preamplifier and then transmitted to a signal processing instrument， and then converted into Spikes by Autospike software （Autospike v.3.7， Syntech， Hilversum， the Netherlands）. The count of Spikes was 1 min after the reaction， and the recognition and intensity of the neuron signals were determined according to the response amplitude， the frequency and the specific response pattern of the feeding deterrent taste neurons[8]. Statistical method In the behavioral tests， the larvae of H. armigera and H. assulta either fed the treated discs or fed the control discs， so the paired t??tests were used to analyze the test data. In the electrophysiological tests， the results of nerve impulse responses to caffeine in the medial sensilla of H. armigera larvae and H. assulta larvae were statistically analyzed by two??sample test. All data were tested using SPSS 17.0 statistical software.
Results and Analysis
Behavioral test results
As shown in Table 2， both the H. armigera larvae and H. assulta larvae were sensitive to caffeine， and H. armigera larvae fed significantly more control discs than the treated discs （P
Electrophysiological test results
The electrophysiological tip??recording results showed that both H. armigera larvae and H. assulta larvae had one caffeine??sensitive feeding deterrent neuron at the medial sensilla. As shown in Fig. 1， the electrophysiological pulse response to 1 and 10 mM caffeine of the medial sensilla of H. assulta larvae were significantly stronger than that of H. armigera larvae， indicating that the feeding deterrent neurons of H. assulta was more sensitive to caffeine than that of H. armigera （P
Conclusion and Discussion
The plant secondary compounds produced by the plant play an important role in the feeding process for both polyphagous and oligophagous insect. There are a variety of secondary compounds that act to inhibit or stop insects from feeding， known as deterrents. Insects have deterrent neurons that can sense the external environment， and the activation of such neurons can inhibit or even stop the feeding behavior of insects[4-5]. Caffeine is a deterrent for many herbivorous insect larvae[6]， and it has been reported that caffeine can cause impulse response of insect deterrent neurons[10]. In this study， we compared the behavior and electrophysiological responses of the polyphagous H. armigera and the oligophagous H. assulta to caffeine. The results show that caffeine is a deterrent for both larvae， but it shows much stronger deterring effects on for oligophagous H. assulta. Both H. armigera larvae and H. assulta larvae have a caffeine??sensitive feeding deterrent neuron at the medial sensilla， and the electrophysiological pulse response of H. assulta larvae is significantly stronger than that of H. armigera larvae. The above results indicate that the response of oligophagous insects to deterrents is stronger than that of polyphagous insects， which strongly suggests that during the evolutionary process， oligophagous insects are more sensitive to and much stronger to the deterrents in the environment than polyphagous insects. It is more beneficial for the former to search for specific host plants. On the other hand， the relatively low susceptibility of polyphagous insects to deterrents may be related to the fact that they feed on multiple host plants， a property that may be beneficial for their adaptation and survival. In addition to the medial sensilla of H. armigera， which have deterrent neurons， the lateral sensilla also have the neurons that are sensitive to caffeine[11]. Some scholars in China have also found that the lateral sensilla of H. armigera， larvae react strongly to azadirachtin[12]. However， since no caffeine??sensitive neurons are found in the lateral sensilla of H. assulta， this study only compares the impulse responses to caffeine in the medial sensilla of H. armigera larvae and H. assulta larvae. Although the results can explain the response of behavioral tests， the effects of lateral sensilla and other sensilla cannot be ruled out. For example， the maxillary palp of H. assulta larvae have the neurons sensing deterrents[13]. The chemical information received by these sensilla is integrated in the central nervous system to reflect behavioral feeding or feed deterring， which is a very complicated process.
References
[1] WU KM， GUO YY. The evolution of cotton pest management practices in China[J]. Annual Review of Entomology， 2005， 500： 31-52.
[2] WANG CZ， DONG JF. Interspecific hybridization of Helicoverpa armigera and H. assulta （Lepidoptera： Noctuidae）[J]. Chinese Science Bulletin 2001， 46（6）： 489-491.
[3] MILES SC， DEL CAMPO ML. Behavioral and chemosensory responses to a host recognition cue by larvae of Pieris rapae[J]. Journal of Comparative Physiology. A， 2005， 191： 147-155.
[4] SCHOONHOVEN LM， VAN LOON JJA. An inventory of taste in caterpillars： Each species its own key[J]. Acta Zoologica Academiae Scientiarum Hungaricae， 2002， 48： 215-263.
[5] DETHIER VG. The role of taste in food intake： A comparative view[M]. In Simon SA and Roper SD （eds.） Mechanisms of Taste Transaction. Boca Raton： FL.CRC. 1993：3-25.
[6] BERNAYS EA， CHAPMAN RF. Taste cell responses in the polyphagous arctiid， Grammia geneura： towards a general pattern for caterpillars[J]. Journal of Insect Physiology， 2001， 47： 1029-1043.
[7] TANG QB， JIANG JW， YAN YH， et al. Genetic analysis of larval host??plant preference in two sibling species of Helicoverpa[J]. Entomologia Experimentalis Et Applcata， 2006，118： 221-228.
[8] ZHOU DS， WANG CZ， VAN LOON JJA. Chemosensory basis of behavioral plasticity in response to deterrent plant chemicals in the larva of the Small Cabbage White butterfly Pieris rapae[J]. Journal of Insect Physiology， 2009， 55： 788- 792.
[9] HODGSON ES， LETTVIN JY， ROEDER KD. Physiology of a primary receptor unit[J]. Science， 1955， 122： 417-418.
[10] GLENDINNING JI， ENSSLEN S， EISENBERGL ME， et al. Diet??induced plasticity in the taste system of an insect： localization to a single transduction pathway in an identified taste cell[J]. Journal of Experimental Biology， 1999， 202： 2091-2102.
[11] ZHANG HJ， FAUCHER PC， ANDERSON A. Comparisons of contact chemoreception and food acceptance by larvae of polyphagous Helicoverpa armigera and oligophagous Bombyx mori[J]. Journal of Chemical Ecology， 2013， 39： 1070-1080.
[12] TANG DL， WANG CZ， LUO LE， et al. Comparison of the reaction characteristics of some compounds of Helicoverpa armigera and Helicoverpa assulta[J]. Science in China （Series C）， 2000， 30（5）： 511-516.
[13] GLENDINNING JI， VALCIC S， TIMMERMANN BN. Maxillary palps can mediate taste rejection of plant allelochemics by caterpillars[J]. Journal of Comparative Physiology. A， 1998， 183： 35-43.
Key words Helicoverpa armigera； H. assulta； Caffeine； Feeding deterrent； Tip??recording
Helicoverpa armigera is a worldwide agricultural pest[1]. It is also a typical polyphagous insect. The host plants include more than 200 species in over 30 families like Solanaceae， Leguminosae， and Compositae. However， its closely related species of H. assulta is an oligophagous insect， which only feeds on a few plants such as pepper and tobacco[2]. The two species of insects can detect the soluble taste compounds in the external environment mainly through the taste receptors on maxillary palpus and epipharynx， as well as the medial and lateral sensilla on the Galea[3]. Multiple taste neurons in the sensilla on the lateral sensiilla play a crucial role in feeding behavior[4]. Feeding deterrent neuron can sense the feeding deterrent in external environment， which can inhibit or even stop the feeding behavior of insects[4-5]. Caffeine is a feeding deterrent for many insect larvae[6]， but little is known about its feed deterring effects on H. armigera and H. assulta. This study compared the behavior and electrophysiological responses of the polyphagous H. armigera and the oligophagous H. assulta to caffeine， which provided a theoretical basis for understanding the mechanism of the taste perception of different feeding insects.
Materials and Methods
Materials
Plants and insects H. armigera and H. assulta were collected from the field and cultured in the laboratory for the second generation at the temperature of （25??1）??， relative humidity of 60%-70%， and photoperiod of L16： D8. After 3 instars old， the larvae were kept in the glass worm??culturing tubes， one per tube， and artificial diet was placed in the tubes. The tubes were blocked with cotton plugs. Adults were placed in cages and fed with 10% honey water. The larvae used for behavioral experiments and electrophysiological tests were reared to 4 instars normal artificial diet. The larvae were taken out from the insect tubes to at molting stage of the 4th instar and placed in empty tubes. After the larvae reached the 5 instars， they were starved for about 8 h. Preparation of feed The artificial feed formula for H. armigera and H. assulta is shown in Table 1.
Test compound and behavioral experimental medium Caffeine was purchased from Sigma??Aldrich with a purity of >99%. The behavioral experiment used whatman?k glass fiber filter paper as the feeding medium and was purchased from Sigma??Aldrich.
Methods
Behavioral test Behavioral tests were performed using dual??choices tests with glass fiber filter paper as the feeding medium. The fiber paper was punched into discs with the diameter of 1 cm. Then， 10 ??l of 10 mM caffeine solution was evenly applied to the glass fiber filter discs with a pipette， which were the treated discs， and 10 ??l of distilled water was evenly applied to other glass fiber filter discs， served as the control discs. The starved 5??instar larvae were placed in the middle of the petri dish and the tests were carried out in a greenhouse at 25 ??. Then， 2 treated filter discs and 2 control filter discs were placed on the inner edge of the petri dish sequentially[7]， and when about 50% of any of the treated discs or control discs were fed， the area of the remaining discs was scanned with an HP scanner and the surface area was calculated using Scion Image software. And the Feeding Deterrent Index （FDI） was calculated. FDI indicated that the intensity of the feeding deterrent deterring the feeding， and it was calculated using the following formula：
FDI=100 （CT）/（C+T）
Where， C indicates that feeding area of the control disc； T is the feeding area of the treated disc. The value of FDI was between 1-100， and the larger value suggested a stronger feeding deterring effect[8].
Electrophysiological test The neuro??pulse response of caffeine in the lateral and medial sensilla was tested using the electrophysiological Tip??recording method[9]. The larvae of H. armigera and H. assulta at the beginning of 5 instars were taken， and then their heads were cut quickly with a scalpel blade. Then， the head was put at one end of the control electrode， which was slightly tilt， and the lower jaw was strutted to outspread the lateral and medial sensilla. The other end of the control electrode was connected to a preamplifier （Syntech Taste Probe DTP??1， Hilversum， the Netherlands）， which was connected to a signal processing instrument （A/D??interface， Syntech IDAC??4， Hilversum， the Netherlands） together. Caffeine was dissolved in 2 mM KCl solution， which was the control solution of the electrophysiological test， and the caffeine solution of 0.1， 1 and 10 mM was made separately. The caffeine solution was then injected into the glass micropipette with a tip diameter of about 30 ??m， which was the test electrode. During the test， the nerve impulse signals of the taste neuron reaction were amplified by a preamplifier and then transmitted to a signal processing instrument， and then converted into Spikes by Autospike software （Autospike v.3.7， Syntech， Hilversum， the Netherlands）. The count of Spikes was 1 min after the reaction， and the recognition and intensity of the neuron signals were determined according to the response amplitude， the frequency and the specific response pattern of the feeding deterrent taste neurons[8]. Statistical method In the behavioral tests， the larvae of H. armigera and H. assulta either fed the treated discs or fed the control discs， so the paired t??tests were used to analyze the test data. In the electrophysiological tests， the results of nerve impulse responses to caffeine in the medial sensilla of H. armigera larvae and H. assulta larvae were statistically analyzed by two??sample test. All data were tested using SPSS 17.0 statistical software.
Results and Analysis
Behavioral test results
As shown in Table 2， both the H. armigera larvae and H. assulta larvae were sensitive to caffeine， and H. armigera larvae fed significantly more control discs than the treated discs （P
Electrophysiological test results
The electrophysiological tip??recording results showed that both H. armigera larvae and H. assulta larvae had one caffeine??sensitive feeding deterrent neuron at the medial sensilla. As shown in Fig. 1， the electrophysiological pulse response to 1 and 10 mM caffeine of the medial sensilla of H. assulta larvae were significantly stronger than that of H. armigera larvae， indicating that the feeding deterrent neurons of H. assulta was more sensitive to caffeine than that of H. armigera （P
Conclusion and Discussion
The plant secondary compounds produced by the plant play an important role in the feeding process for both polyphagous and oligophagous insect. There are a variety of secondary compounds that act to inhibit or stop insects from feeding， known as deterrents. Insects have deterrent neurons that can sense the external environment， and the activation of such neurons can inhibit or even stop the feeding behavior of insects[4-5]. Caffeine is a deterrent for many herbivorous insect larvae[6]， and it has been reported that caffeine can cause impulse response of insect deterrent neurons[10]. In this study， we compared the behavior and electrophysiological responses of the polyphagous H. armigera and the oligophagous H. assulta to caffeine. The results show that caffeine is a deterrent for both larvae， but it shows much stronger deterring effects on for oligophagous H. assulta. Both H. armigera larvae and H. assulta larvae have a caffeine??sensitive feeding deterrent neuron at the medial sensilla， and the electrophysiological pulse response of H. assulta larvae is significantly stronger than that of H. armigera larvae. The above results indicate that the response of oligophagous insects to deterrents is stronger than that of polyphagous insects， which strongly suggests that during the evolutionary process， oligophagous insects are more sensitive to and much stronger to the deterrents in the environment than polyphagous insects. It is more beneficial for the former to search for specific host plants. On the other hand， the relatively low susceptibility of polyphagous insects to deterrents may be related to the fact that they feed on multiple host plants， a property that may be beneficial for their adaptation and survival. In addition to the medial sensilla of H. armigera， which have deterrent neurons， the lateral sensilla also have the neurons that are sensitive to caffeine[11]. Some scholars in China have also found that the lateral sensilla of H. armigera， larvae react strongly to azadirachtin[12]. However， since no caffeine??sensitive neurons are found in the lateral sensilla of H. assulta， this study only compares the impulse responses to caffeine in the medial sensilla of H. armigera larvae and H. assulta larvae. Although the results can explain the response of behavioral tests， the effects of lateral sensilla and other sensilla cannot be ruled out. For example， the maxillary palp of H. assulta larvae have the neurons sensing deterrents[13]. The chemical information received by these sensilla is integrated in the central nervous system to reflect behavioral feeding or feed deterring， which is a very complicated process.
References
[1] WU KM， GUO YY. The evolution of cotton pest management practices in China[J]. Annual Review of Entomology， 2005， 500： 31-52.
[2] WANG CZ， DONG JF. Interspecific hybridization of Helicoverpa armigera and H. assulta （Lepidoptera： Noctuidae）[J]. Chinese Science Bulletin 2001， 46（6）： 489-491.
[3] MILES SC， DEL CAMPO ML. Behavioral and chemosensory responses to a host recognition cue by larvae of Pieris rapae[J]. Journal of Comparative Physiology. A， 2005， 191： 147-155.
[4] SCHOONHOVEN LM， VAN LOON JJA. An inventory of taste in caterpillars： Each species its own key[J]. Acta Zoologica Academiae Scientiarum Hungaricae， 2002， 48： 215-263.
[5] DETHIER VG. The role of taste in food intake： A comparative view[M]. In Simon SA and Roper SD （eds.） Mechanisms of Taste Transaction. Boca Raton： FL.CRC. 1993：3-25.
[6] BERNAYS EA， CHAPMAN RF. Taste cell responses in the polyphagous arctiid， Grammia geneura： towards a general pattern for caterpillars[J]. Journal of Insect Physiology， 2001， 47： 1029-1043.
[7] TANG QB， JIANG JW， YAN YH， et al. Genetic analysis of larval host??plant preference in two sibling species of Helicoverpa[J]. Entomologia Experimentalis Et Applcata， 2006，118： 221-228.
[8] ZHOU DS， WANG CZ， VAN LOON JJA. Chemosensory basis of behavioral plasticity in response to deterrent plant chemicals in the larva of the Small Cabbage White butterfly Pieris rapae[J]. Journal of Insect Physiology， 2009， 55： 788- 792.
[9] HODGSON ES， LETTVIN JY， ROEDER KD. Physiology of a primary receptor unit[J]. Science， 1955， 122： 417-418.
[10] GLENDINNING JI， ENSSLEN S， EISENBERGL ME， et al. Diet??induced plasticity in the taste system of an insect： localization to a single transduction pathway in an identified taste cell[J]. Journal of Experimental Biology， 1999， 202： 2091-2102.
[11] ZHANG HJ， FAUCHER PC， ANDERSON A. Comparisons of contact chemoreception and food acceptance by larvae of polyphagous Helicoverpa armigera and oligophagous Bombyx mori[J]. Journal of Chemical Ecology， 2013， 39： 1070-1080.
[12] TANG DL， WANG CZ， LUO LE， et al. Comparison of the reaction characteristics of some compounds of Helicoverpa armigera and Helicoverpa assulta[J]. Science in China （Series C）， 2000， 30（5）： 511-516.
[13] GLENDINNING JI， VALCIC S， TIMMERMANN BN. Maxillary palps can mediate taste rejection of plant allelochemics by caterpillars[J]. Journal of Comparative Physiology. A， 1998， 183： 35-43.