J Insect Behav (2011) 24:175–185 DOI 10.1007/s10905-010-9246-4
Side-Dominance of Periplaneta americana Persists Through Antenna Amputation Rodrigo Cooper & Nicholas Nudo & Jorge M. González & S. Bradleigh Vinson & Hong Liang
Revised: 27 August 2010 / Accepted: 13 October 2010 / Published online: 28 October 2010 # Springer Science+Business Media, LLC 2010
Abstract While brain lateralization is widely studied for vertebrates, only a few studies have been performed on insects. In the present research, we investigated the behavior of the common American cockroach, Periplaneta americana, to determine if such lateralization is evident through their decision making process. The roaches were allowed to run through a y-tube and make a decision on which direction to take. Vanilla and ethanol were randomly placed at the ends of the y-tube to entice the roaches to reach the end of the tubes. Tests were repeated by severing one antenna on each roach in different configurations which were expected to alter the decision of the insect. Through a simple statistical analysis we determined that the odors of vanilla and ethanol play an insignificant role in the decision making. We found that injury to the antenna indeed affected their decision, However, similar injury to either antenna showed an innate bias for turning right. Our research supports the hypothesis that Periplaneta americana is right-side dominated in their tactile and odor senses. Keywords Side-preference . Periplaneta americana . y-tube . antenna
The insect antenna is a highly complex and sensitive organ (Kuwana et al. 1995). The antennae of the American cockroach, Periplaneta americana (Linnaeus) (Blattodea: Blattidae), have been studied for their functions as mechanical and chemical receptors (Morita 1959; Morita and Yamashita 1959). Their antennae surfaces are covered with small setae which are mechano- and chemo- receptors that R. Cooper : N. Nudo : H. Liang (*) Department of Mechanical Engineering, Texas A&M University, MS 3123 TAMU, College Station, TX 77843, USA e-mail:
[email protected] J. M. González : S. B. Vinson Department of Entomology, Texas A&M University, College Station, TX 77843, USA
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provide the cockroach information concerning the environment. A large network of nerve fibers from the antennal proprioceptive and exteroceptive organs extends through the antenna to the brain (Schaller 1978; Seelinger and Tobin 1981). In general, Periplaneta americana roaches show an affinity to human constructions and dwellings and live in places that can easily provide water, food, shelter and warmth. They do most of their activities during the night. They are also cursorial, which means that they are adapted to run having a rate of locomotion of 750–1,400 mm per second at 25°C. When resting they do it in crevices during the day often in association with other roaches. Gregarious insects like cockroaches depend on olfactory senses to locate other individuals of its own group as well as tracking chemical cues important to food location (Waldow and Sass 1984; Jeanson and Deneubourg 2006). Olfactory sensitivity also appears to depend on wind conditions (Willis and Avondet 2005) and on the type of chemical being sensed. For example, alcohols from rotting food elicit a lower stimulus response from sexually active males than female pheromones (Wharton et al. 1962; Boeckh et al. 1963; Getz and Akers 1997a,b). Injury to the flagellum can cause a decrease in the ability of P. americana to detect or smell chemicals. While many insects can use their antennae as taste sensor, no such gustatory ability has been detected in the antennae of P. americana (Schneider 1964). Long antennae on many insects have apparently evolved to increase the number of chemical receptors for increased sensitivity (Schneider 1964). On close inspection of several insects, including P. americana, the density of the sensilla is too low to qualify as a high sensitivity sensor. Having such a long antenna without taking full advantage of its surface with more sensors may be explained by employing the antenna as tactile organs. Mechanoreceptors in the antenna are used for detecting mechanical perturbations, such as wind perturbations, and as a tactile guidance system. Touching the antennae cause a quick escape response in static insects. P. americana uses its antennae as a tactile means of guidance and obstacle avoidance while performing escape maneuvers. During an escape run, P. americana drags one antennal flagellum along a wall while maintaining the pedicel at nearly a constant angle relative to the body (Cowan et al. 2006). This dragging of the flagellum provides the feedback response required to maintain constant distance from walls or other objects (Camhi and Johnson 1999). Escape maneuvers require constant contact with the wall while slower walking allows the roach to break contact with the wall and explore its surroundings. The importance of the antenna as a means of tactile guidance was studied by (Okada and Toh 2004). They demonstrated that P. americana depended highly on the tactile cues of the antennae when its optical senses were inhibited. Okada covered the eyes with wax and carbon black and placed the roaches in an open arena. When the blind cockroach encountered a static object, it would examine it with repeated antennal contacts, finally approaching and coming to rest next to the object. Left and right side dominance is not as widely studied in invertebrates such asinects. It has been witnessed in vertebrates, such as Bengalese finches, that a distinctive side preference in responding to singing calls was observed (Okanoya et al. 2001). It is believed that most lower invertebrates, birds, and mammals demonstrate lateralization due to a homologous trait shared by a common ancestor (Vallortigara et al. 1999). However, there are studies indicating that bumblebees
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(Rowell 1964; Kells and Goulson 2001) have a side preference in their inflorescence visits or approaches while honeybees have a lateralization for ease of learning and memory recollection (Letzkus et al. 2006; Louis et al. 2008; Frasnelli et al. 2010). Furthermore, tests on fruit flies indicate that an asymmetric brain is necessary in order to generate long-term memory (Pascual et al. 2004; Duistermars et al. 2009). This lead for us to hyposize that most animals with the ability to learn have an asymmetric brain. Other studies also indicated that in an otherwise symmetric brain, lateralization occurs to more effectively process visual cues, particularly in lateral monocular eyes (Bisazza et al. 1998). With both eyes functioning as independent sources of its surroundings, animals with lateral eyes need to process the information simultaneously by one common part of the brain in order to avoid conflicting responses. Lateralization also aids in avoiding redundant duplication of similar brain functions (Vallortigara and Rogers 2005). The brain of P. americana is symmetrical in structure. In this research we focus on P. americana to determine if their behavior can indicate a level of lateralization shown in other insects. Our team of entomologists and engineers have been working together to develop ways to track roach movements and the data we had gathered suggested there might be a bias to move more in one direction than another.
Materials and Methods Insects Tests were conducted using the common American cockroach, P. americana. These insects are reared and maintained in our laboratory in plastic containers provided with food (Dog food, Puppy Chow, Inc.) and water ad libitum. Dead insects are removed as soon as they are found in order to prevent disease and contamination. Colonies are kept at temperature between 20°C and 25°C and alternating light and dark cycles of 12 h each. All insects used in this experiment were adults and special attention was paid to ensure equal number of males and females tested. Thirty-eight roaches were tested for each of the five conditions: whole antennae, half the left antenna cut, whole left antenna cut, half the right antenna cut, and whole right antenna cut. Half-antenna amputations were performed to observe if there was an intermediate effect on the reaction of the insects. Full-antenna amputations were performed to determine if both antennae were used equally during walking in low visibility conditions. If full antenna amputation of either antenna resulted in the same percentage of ipsilateral tube decisions, then both antennae would be equally biased. Details on each of the conditions and preparations are described in a following section. Selection of Chemicals Two chemicals with distinct odors, vanilla extract (McCormick, Inc.) and ethanol were used in the experiments. A cotton ball was immersed to saturation in each of the chemicals to be tested and placed at the end of each of the y-tube’s arms. Undiluted vanilla extract was chosen due to its demonstrated appeal (Balderrama 1980). Due to the insects’ noted preference to associate with trash areas containing
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rotten foods, ethanol (Sygma-Aldrich, 99.5% pure) was used as the second chemical since it is a byproduct of decaying fruits. All tests were performed with both scents placed at one of the ends of the y-tube. The location of each scent was randomized while special precaution was taken to ensure that each scent was placed on both tubes the same number of trails. Y-tube Design P. americana is known to produce a waxy coating on the cuticle (Beament 1955; Cooper et al. 2009). This wax has, among many other uses, the ability to produce a pheromone scent trail (Boeckh et al. 1963) that can be detected by other co-specific individuals (Jeanson and Deneubourg 2006). This sent can influence their decisionmaking process. The male P. americana has a higher number of olfactory receptors (Bell 1982) which makes them more sensitive to those pheromones. For the above mentioned reason a y-tube with a replaceable bottom was made to prevent any chemical influence from previously tested P. americana. The y-tube channels have a square cross-section of 100×100 mm made of Plexiglas 5 mm thick. The bottomless y-tube was placed on a flat surface covered with butcher paper (Office Depot®). The paper was changed at the end of each test. Furthermore, the interior walls of the channels were coated with Vaseline® to prevent insects from walking on the walls. The y-tube and its dimensions are shown in Fig. 1. End Caps Special end caps were designed to allow handling the P. americana and allow air flow into the y-tube. The end of the y-tube was fitted with an adaptor for an entrance
Fig. 1 The Y-tube configuration with entrance, exit traps, and scent sources
Scent1
Scent2
Trap
Trap
30.5 cm
.3
20 cm
40.6 cm
10
Entrance
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cage. The cage is a cylinder of polystyrene with a 25 mm diameter hole open on the side that was inserted into the y-tube. The hole allows the introduced insect to exit the cage towards the y-tube. Once the insect crossed this hole into the tube the experimental time commenced. The two choice ends of the y-tube were fitted with a similar apparatus to permit easy removal of the cage and scent holder. Cylinders of the same type as previously described were used as the exit cage and scent holders. The first cylinder from the y-tube extremity is a trapping cage. The roach entered the cylinder through a round hole (25 mm) at the center of the cylinder. Once the insect entered this trapping cage, the experimental time ended. The second cylinder contained the scent chemical. A round hole with a wire mesh allowed the scent to enter the exit cage and y-tube but kept the insect from directly contacting the chemical. Choice Selection All tests were performed in a dark room with the lights off with the exception of a soft reading light placed equidistant from both exit channels and over the entrance channel. To test for choice preference, each P. americana was tested individually. Thirty-eight roaches were used to test for each condition. Each insect was allowed to complete the y-tube only once to prevent habituation. In many instances the roaches failed to exit the entrance chamber or make a selection once in the y-tube. Roaches were returned to the colony box if they did not enter the exit cage after 10 min. In most cases, the insects entered the exit cage within 3 min. On rare occasions when the test insect failed to select one of the arms of the y-tube after 5 min, it was removed and returned to the colony box. To limit repetition of a failed run, each insect was limited to attempt the tube no more than twice a day and no more than 4 times per week until a successful selection was made. Upon completion of a successful run, the roach was placed in a separate container and not reused in further tests. Roaches tested in one condition were not reused in other conditions to avoid any effects of habituation that may have occurred during their first attempt. Initially, the hole in the entrance cage is closed with a rubber stopper. An individual P. americana was placed in the entrance cage and a rubber stopper is placed only for transport. The startled insect was kept in the cage for 5 min. That was enough time for the roach to resume a resting or slow walking rhythm indicating that it was accustomed to the cage prior to releasing into the y-tube. The rubber stopper was removed and the cage inserted into the single-ended channel. A fan from a used computer that pulls air was fitted on the entrance cage that extracted air from the y-tube, forcing air over the chemicals and into the y-tube at a rate of 5CFM. Special care was taken to make the holes on the cages of the same size to provide a uniform airflow through both channels. Once the insect entered the exit cage, the coupled cage and scent holder were removed. A rubber stopper was placed on the hole of the exit cage to prevent the insect from escaping during return to the rearing chamber. Prior to every third test the y-tube was rotated 180° in order to control for any variables from the room such as disparities in air flow, discrepancies in lighting, and other environmental effects.
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Antenna Amputation Testing Two forms of antennal amputation were performed on CO2 anesthetized individuals— cutting off the distal half and complete antennectomy. Anesthesia of the insects during amputation is commonly performed to reduce trauma and facilitate handling (Eisemann et al. 1984). Similar damage occurs naturally for roaches raised in the wild. However, the insects were kept in the rearing box since there was no need for euthanization since they are able to adapt to mutilations and cannot feel pain (Wigglesworth 1980). For the half-antenna amputation, the length of the antenna was first measured and then cut in half using high precision microsurgical scissors. The whole antenna amputation was performed by cutting the antenna at the joint between the scape and the pedicel (Fig. 2). After amputation, the insects were allowed to fully recover for a minimum of 30 min prior to replacement in the y-tube. Statistical Methods Due to the number of variables presented during these experiments, a statistical analysis was performed to identify what factors were of highest importance to the outcome and which could be neglected. Since it is known that the antennae of P. americanaare used for both tactile and scent detector, it is necessary to verify which of the two senses played a great role in path determination. The dependence of P. americana’s behavior on its antennae makes them an excellent subject for study of side preference.
Flagellum
Pedicel Scape
Fig. 2 Scanning electron microscope image showing the head of a P. americana with a completely severed right antenna
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The statistical analysis was conducted using the Null-hypothesis theorem(Mason et al. 2003), which may be presented as an equation: Y ¼ bX þ " where β is a scaling factor, matrix X is the matrix form of the test conditions, ε is the error (neglected in this case), and Y is the matrix form of the results. The {X} matrix is composed of three conditions: {X1}= side of antenna cut, {X2} = amount of that antenna cut, and {X3} = location of scents with respect to the tube entrance. Each individual matrix is composed by providing numerical values to given cases of the variable defined by that matrix. For example, {X1} is composed by giving cases with a left antenna cut a value of 1 and cases with a right antenna cut a value of 2. Similarly, the {Y} matrix is formed by giving a value to the selection of the roach. The only matrix that is unknown is the β factors. The higher the value of β, the higher the relevance it has towards the outcome of the result. However, the values do not determine a quantifiable level of dependence, simply a relative value of dependence amongst the other variables. An exact binomial test was performed on the data to determine the reliability of the conclusions. The null hypothesis was that all insects with both antennae intact would choose both sides of the y-tube in an equal ratio, a roach with a whole antenna removed would go to the opposite side all the time, and a roach with half of an antenna would go to the opposite side three quarters of the time. A P-value for a two-tailed case is obtained. A second exact binomial test was performed to determine the outcome of the scent selection. The null hypothesis predicts that all roaches will choose both scents equally.
Results and Discussion Allowing the roach to relax in the entrance cage prior to releasing into the y-tube prevented an alarmed escape behavior through the channels. Once the air flow was initiated and the rubber stopper removed, the roaches investigated the entrance cage thoroughly, finding the exit hole within 3 min. The roaches then proceeded to venture into the main channel inspecting both walls but unable to climb them. The probabilities obtained through experiments are shown in Tables 1 and 2. For the case where roaches have both intact antennae, 57% of the roaches preferred taking the right path. Roaches with the entire left antenna cut chose the right path 93% of the time, whereas roaches with the entire right antenna removed only went left 70% of the time. Similarly, roaches with half of a left prefer the right path 71% of the trials, where roaches with half a right antenna preferred the left path only 59%. The roach has a higher tendency to turn right when the left antenna is damaged, and a lower tendency to turn left when the right antenna is damaged. Results are summarized in Fig. 3. This would imply a bias to turn right regardless of damage to the antennae. Since the treatments were randomized in the y-tube tests external factors are considered negligible. As mentioned earlier, the antenna of a P. americana is highly sensitive to smell and when damaged, sensitivity is reduced. The odors from the chemicals at the end
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Table 1 Exact fit P-test verifies that roaches will follow the predicted outcome with high reliability, except for the roaches with the entire right antenna cut. The strong rejection of the null hypothesis is evidence that the roaches are laterally dominant on the right side Antenna cut condition
Prediction
P-value (two-tailed)
Left 100%
1:0 (right)
0.029207
Left 50%
3:1 (right)
0.664074
None
1:1
0.663624
Right 50%
1:3 (left)
0.073519
Right 100%
0:1 (left)
1.73E-10
of the channels were placed for two reasons: to motivate the roach to travel through the channels and to determine if injury would affect its use of its sense of smell. Figure 4 shows that scent location had no effect on path choice. The probability of roaches choosing one scent over another ranged between 50% and 54%. It is interesting to mention that only when the entire right antenna was amputated did these choices maintain any real effect, with the roaches following the ethanol scent in 70% of the trials. The statistical analysis was performed to determine to different factors played into the decision-making process of the roach. By using the null-hypothesis method and solving for the scaling factor β. The values of β where taken for the side of the antenna cut, the amount of the antenna cut, and the location of the scent. From our experiments, we determined these values to be [β]=[0.5819 0.3175 0.2120]. This indicates that the path selection process is dominated by which antenna is cut, followed by the amount of the antenna cut, and scent location least important. An exact binomial test for goodness of fit of the data is compared to the initial predictions previously encountered. P-values for a two-tailed result are presented in Table 1. The values indicate that the null hypothesis is supported, except for the case of the whole right antenna amputation. This indicates that while it is safe to assume that the majority of roaches with the left antenna amputated will go right, the same is not necessarily true if the entire right antenna is removed. A similar exact binomial test was performed for the data comparing the scent selection. In this case, since the antennae do not have many chemoreceptors, it is
Table 2 Exact fit P-test verifies that scent selection was not a determining factor for the path selection of the roaches Antenna cut condition
Prediction
P-value (two-tailed)
Left 100%
1:1
0.701108038
Left 50%
1:1
1
None
1:1
1
Right 50%
1:1
0.122078121
Right 100%
1:1
0.35055
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Fig. 3 Probabilities of choices taken by Periplaneta americana in their paths. In all conditions, the roach has a higher tendency to take the right path. Most notably, roaches with no left antenna will go right about 93% of the time, while roaches with no right antenna will go left only about 70% of the time
believed that the selection will not be affected by scent. Indeed, as shown in Table 2, there is no significant deviance from the prediction to declare it incorrect. In all testing conditions with opposite antenna cut the same length, it is seen that the right path predominates in the selection process of the roaches. Thus there appears to be an uneven effect caused by equal amputation of the different antennae. In addition, when the entire right antenna is cut, the roach will tend to follow the more volatile scent, ethanol This could be due to its increased use of other senses in the absence of its ‘dominating’ antenna, much like humans compensate a loss of one sense with increased perception of other senses. In roaches, this preference in the right path selection and choice of ethanol only in the absence of the entire right antenna can be attributed to having dominance in sensing on the right antenna. Since the antenna is the main method of investigatory and escape motions, we explore the notion that the roach is effectively right-handed, or in the case of P. americana, right-antennaed.
Fig. 4 Probabilities of choices made by Periplaneta americana under the influence of smell. In general, antennaantenna cut seems to have no effect on smell choices. However, whenWhen the “stronger” antenna was cut (100% right), the insect prefers ethanol over vanilla
56% 56%
56%
56%
56%
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Conclusions Handedness in roaches was demonstrated through a series of y-tube experiments. When comparing the results for when the entire antenna was cut from either side, the roaches had a higher tendency for turning right than left. Similar results were obtained when either antenna was cut in half. More evident was the skew towards the right path when both antennae were fully intact. Future research will focus on the detailed study in the side dominance related to appendages of the roach body.
Acknowledgements The research was supported by the NSF 0515930. The authors thank Ms. S. Jill Butler from Industrial Engineering at Texas A&M University for assistance in collecting and analyzing the data. We are indebted to Robert W. Matthews (University of Georgia) for his enlightening comments. SEM image collected by Hyungoo Lee at the Texas A&M University was greatly acknowledged.
References Balderrama N (1980) One trial learning in the american cockroach, periplaneta americana. J Insect Physiol 26:499–504 Beament JWL (1955) Wax secretion in the cockroach. J Exp Biol 32:514–538 Bell WJ (1982) The american cockroach Bisazza AJ, Rogers L, Vallortigara G (1998) The origins of cerebral asymmetry: a review of evidence of behavioural and brain lateralization in fishes, reptiles and amphibians. Neurosci Biobehav Rev 22:411–426 Boeckh J, Priesner E, Schneider D, Jacobson M (1963) Olfactory receptor response to the cockroach sexual attractant. Science 141:716–717 Camhi JM, Johnson EN (1999) High-frequency steering maneuvers mediated by tactile cues: Antennal wall-following in the cockroach. J Exp Biol 202:631–643 Cooper R, Lee H, Gonzalez JM, Butler J, Vinson SB, Liang H (2009) Lubrication and surface properties of roach cuticle. J Tribol 131:014502 Cowan NJ, Lee J, Full RJ (2006) Task-level control of rapid wall following in the american cockroach. J Exp Biol 209:1617–1629 Duistermars BJ, Chow DM, Frye MA (2009) Flies require bilateral sensory input to track odor gradients in flight. Curr Biol 19:1301–1307 Eisemann CH, Jorgensen WK, Merritt DJ, Rice MJ, Cribb BW, Webb PD, Zalucki MP (1984) Do insects feel pain?—a biological view. Cell Mol Life Sci 40:164–167 Frasnelli E, Vallortigara G, Rogers LJ (2010) Response competition associated with right-left antennal asymmetries of new and old olfactory memory traces in honeybees. Behav Brain Res 209:36–41 Getz WM, Akers RP (1997a) Coding properties of peak and average response rates in american cockroach olfactory sensory cells. Biosystems 40:55–63 Getz WM, Akers RP (1997b) Response of american cockroach (periplaneta americana) olfactory receptors to selected alcohol odorants and their binary combinations. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 180:701–709 Jeanson R, Deneubourg J-L (2006) Path selection in cockroaches. J Exp Biol 209:4768–4775 Kells AR, Goulson D (2001) Evidence for handedness in bumblebees. J Insect Behav 14:47–55 Kuwana Y, Shimoyama I, Miura H (1995) Steering control of a mobile robot using insect antennae. In: Shimoyama I (ed) Intelligent robots and systems 95. ‘Human robot interaction and cooperative robots’, Proceedings. 1995 IEEE/RSJ International Conference on, vol. 2. pp 530–535 vol. 2 Letzkus P, Ribi WA, Wood JT, Zhu H, Zhang S-W, Srinivasan MV (2006) Lateralization of olfaction in the honeybee apis mellifera. Curr Biol 16:1471–1476 Louis M, Huber T, Benton R, Sakmar TP, Vosshall LB (2008) Bilateral olfactory sensory input enhances chemotaxis behavior. Nat Neurosci 11:187–199 Mason RL, Gunst RF, Hess JL (2003) Statistical design and analysis of experiments: with applications to engineering and science. Wiley, Hoboken
J Insect Behav (2011) 24:175–185
185
Morita H (1959) Initiation of spike potentials in contact chemosensory hairs of insects. Iii. D.C. Stimulation and generator potential of labellar chemoreceptor of calliphora. J Cell Comp Physiol 54:189–204 Morita H, Yamashita S (1959) Generator potential of insect chemoreceptor. Science 130:922 Okada J, Toh Y (2004) Antennal system in cockroaches: a biological model of active tactile sensing. Int Congr Ser 1269:57–60 Okanoya K, Ikebuchi M, Uno H, Watanabe S (2001) Left-side dominance for song discrimination in bengalese finches (lonchura striata var. Domestica). Anim Cogn 4:241–245 Pascual A, Huang K-L, Neveu J, Preat T (2004) Neuroanatomy: brain asymmetry and long-term memory. Nature 427:605–606 Rowell CHF (1964) Central control of an insect segmental reflex: I. Inhibition by different parts of the central nervous system. J Exp Biol 41:559–572 Schaller D (1978) Antennal sensory system of periplaneta americana l. Cell Tissue Res 191:121–139 Schneider D (1964) Insect antennae. Annu Rev Entomol 9:103–122 Seelinger G, Tobin T (1981) Sense organs. In: The american cockroach. Chapman and Hall, London, pp 217–245 Vallortigara G, Rogers LJ (2005) Survival with an asymmetrical brain: advantages and disadvantages of cerebral lateralization. Behav Brain Sci 28:575–589 Vallortigara G, Rogers LJ, Bisazza A (1999) Possible evolutionary origins of cognitive brain lateralization. Brain Res Rev 30:164–175 Waldow U, Sass H (1984) The attractivity of the female sex pheromone ofperiplaneta americana and its components for conspecific males and males ofperiplaneta australasiae in the field. J Chem Ecol 10:997–1006 Wharton DRA, Black ED, Merritt C Jr, Wharton ML, Bazinet M, Walsh JT (1962) Isolation of the sex attractant of the american cockroach. Science 137:1062–1063 Wigglesworth VB (1980) Do insects feel pain? Antenna 1:8–9 Willis MA, Avondet JL (2005) Odor-modulated orientation in walking male cockroaches periplaneta americana, and the effects of odor plumes of different structure. J Exp Biol 208:721–735
