How Females of Lucihormetica Verrucosa Might See the Pronotal Spots of Their Mates 1

How females of Lucihormetica verrucosa might see the pronotal spots of their mates 1

Entomologie heute 30 (2018): 1-8

How Females of Lucihormetica verrucosa (Blattodea, Blaberidae) Might See the Pronotal Spots of Their Mates

Wie Weibchen von Lucihormetica verrucosa (Blattodea, Blaberidae) die Pronotum-Flecken ihrer Partner sehen könnten

JANA BECKERT, HARTMUT GREVEN & KLAUS LUNAU

Zusammenfassung: In Wahlversuchen haben wir Hinweise dafür gewonnen, dass die Weibchen der Leuchtschabe Lucihormetica verrucosa Männchen mit roten Pronotumﬂ ecken, d.h. mit einem hohen Carotinoidgehalt der Epidermis, die unter der transparenten Pronotum-Cuticula liegt, attraktiver ﬁ nden als Männchen mit weißen oder gelben Flecken, deren Epidermis keine oder nur wenige Ca- rotinoide enthält. Wenn man davon ausgeht, dass die Balz bei L. verrucosa zumindest partiell visuell gesteuert wird und dass diese Schabenart als nacht- oder dämmerungsaktive Schabe „rotblind“ ist, aber über UV- und Grünrezeptoren verfügt, stellt sich die Frage, wie die Weibchen die roten Flecken wahrnehmen. Eine Analyse der Fleckenfarbe mit Hilfe der RGB-Skala zeigt, dass die roten Flecken einen geringeren Grünanteil haben. Dies könnte die Weibchen befähigen, die unterschiedlich gefärb- ten Flecken zu unterscheiden und so indirekt Auskunft über die Qualität des Partners zu erhalten.

Schlüsselwörter: Leuchtﬂ eckschabe, Carotenoide, Partnerwahl

Summary: Based on choice experiments we found some evidence that males of the cockroach Lucihormetica verrucosa with red pronotum spots, i.e. with a high carotenoid content in the epidermis underlying the transparent spot cuticle, are more attractive to females than males with white or yellow spots, whose epidermis does not contain or contains very little amounts of carotenoids. As- suming that courtship of L. verrucosa is at least in part visually triggered and that this nocturnal or crepuscular species is “red-blind”, but has UV and green receptors, the question arises how females might perceive the red spots. An analysis of the spot colour using the RGB scale showed that the red spots have a lower amount of reﬂ ected green light, which may enable females to differentiate between variously coloured spots to gain indirectly information about the quality of their mates.

Key words: Glow spot cockroach, carotenoids, mate choice

1. Introduction the transparent cuticle of the spots (see GREVEN et al., in prep.) instead of within the Males of glow spot cockroaches (genus Lu- fat body as previously suggested (GREVEN cihormetica) are characterized by two distinct & ZWANZIG 2013; BECKERT et al. 2017), (3) white, yellow or red spots on the pronotum. that these carotenoids are exclusively α- and Earlier studies preferably on Lucihormetica β-carotenoids (see GREVEN et al., in prep.), verrucosa have shown (1) that the colour of (4) that the spots reﬂ ect UV-light (ZOMPRO these spots may vary between red, yellow & FRITZSCHE 1999; VRŠANSKÝ et al. 2012; and white depending on the carotenoid VRŠANSKÝ & DUŠAN 2013; BECKERT et al. content of diet (e.g. FRITZSCHE 2013; BE- 2017), (5) that the intensity of this UV CKERT et al. 2017), (2) that the carotenoids reflection decreases with increasing red are stored mainly in the epidermis below colouration (BECKERT et al. 2017), and (6)

Entomologie heute 30 (2018) 2 JANA BECKERT, HARTMUT GREVEN & KLAUS LUNAU that white but not red spots fl uoresce under virgins females were separated immediately UV-light (BECKERT et al. 2017). after the imaginal moult and were reared Because of the diet-dependent colouration without males. and the particular signifi cance of carotenoids for animals serving as visual and colouring 2.1. Experimental procedures pigments, antioxidants and hormone precur- sors (e.g. BLOUNT & MCGRAW 2008; HEATH Two experiments were carried out. In ex- et al. 2013), it has been concluded that the periment I one virgin female was confronted red colouration of the spots may represent with one male (10 tests with 10 males (I-X) a condition-dependent character giving and 10 virgin females (I-X)) (Tab. 1). males (intrasexual selection) and/or females In experiment II one virgin female could (intersexual selection) information about choose between two males (10 tests with the quality of their rivals or mates (GREVEN 20 males (XI-XXX) and 10 virgin females & ZWANZIG 2013). In addition, based on (XI-XX)) (Tab. 1). the assumption that glow spot cockroaches The males were taken from different breed- are bioluminescent, it was speculated they ing boxes, to exlude that they did know might mimic poisonous bioluminescent click each other before the test. Prior to each beetles (see VRŠANSKÝ et al. 2012; VRŠANSKÝ experiment, the length (front margin of the & DUŠAN 2013; for critical remarks on bio- pronotum to the genital opening) of each luminescence in glow spot cockroaches see male was measured using a caliper gauge. especially MERRITT 2013; see also GREVEN & Care was taken to use similarly sized males. ZWANZIG 2013; BECKERT et al. 2017). To distinguish the animals, they were marked All these suggestions have not been tested on the wings with a white Edding pen. experimentally up to now. In the present In each experiment the cockroaches were note we report on mate choice experiments fi lmed over 30 min from above in an “arena” giving some indirect evidence that “red” (plastic box with a base area of 14 x 7 cm) males of L. verrucosa appear more attractive with an Iphone 6S (Apple) attached to a to females than “white” males and discuss, tripod. The videos were analysed with the how this might be possible, assuming that free program “VLCmedia player”. As a L. verrucosa as other nocturnal cockroaches measure of the male’s attractiveness we used do not have red photoreceptors, but have the time (indicated by the time code) the UV and green receptors (e.g. MAZOKHIN- female needed from the start of the test up PORSHNYAKOV & CHERKASOV 1985; MOTE to climb the male, i.e. the shorter this period, & GOLDSMITH 1970; KOEHLER et al. 1987; the more attractive was the male. HEIMONEN et al. 2016). 2.2. Colour analysis of pronotum spots 2. Material and methods The spots of all males that proved to be Animals came from a breeding stock of the attractive to the females were analysed Biology Department of the Heinrich-Heine by means of RGB values using single University Düsseldorf. Cockroaches were images from the videos and the tool “colour held in various plastic containers at 20-25 oC pipette”(radius 4) of the image-editing pro- and were fed either with carotenoid-enriched gram GIMP 2.8.22 (GNU Image Manipula- or carotenoid-poor diet leading to males tion Program, www.gimp.org). with red spots (“red” males) or males with For that, the “arena” was divided into fi ve yellow or white spots (“white” males) (for slightly overlapping subareas and one photo details see BECKERT et al. 2017). To obtain (from the video sequence) was taken from How females of Lucihormetica verrucosa might see the pronotal spots of their mates 3

Fig. 1: Division of the experimental arena in fi ve subareas (red circles) to assess colouring of the pronotal spots. For further explanations see text. Abb. 1: Aufteilung der Arena in fünf Areale (rote Kreise) zur Beurteilung der Farbe der Pronotum- fl ecken. Weitere Erklärungen im Text. each male in each subarea resulting in fi ve Singapore) for UV and fl uorescence photos. images per male. In the case a male did not Normal photos were dis assembled into the stay in a given subarea during the test, he individual colour channels (red, green, blue) was photographed in the nearest distance and reassembled with the UV-photo accor- from this area. Care was taken that the males ding to the suppos ed receptor equipment of were aligned with the upper left corner of cockroaches (for technical details see LUNAU the arena in the direction of the light source & VERHOEVEN 2017). (Fig. 1). To calculate the colours of the pronotum 2.4. Statistics spots we used the (additive) RGB system that describes a colour through its compo- The correlation between the attractiveness nents, i.e. the primary colours red, green of the males and the colour of the prono- and blue. The RGB values indicate the por- tum spot was shown using Spearman’s rank tions of these colours, red, green and blue correlation (www.socscistatistics.com). For (BYERS 2006; www.itwissen.info). From the this purpose, attractiveness of the males fi ve images of both spots per male RGB- (see above) was sorted in ascending order (1- values were calculated for the primary col- 17) (see Tabl. 3), compared with the values ours and from the two averages obtained per calculated with the RGB model (see above) male another mean was formed. and checked for correlation.

2.3. Photography 3. Results

With the help of false colour photography we 3.1. Mate choice and attractiveness simulated the “cockroach-specifi c” view on the spots. We used a Panasonic Lumix GH-1 Experiment I (1♂:1♀): From the 10 males (Kadoma, Japan) modifi ed for false colour fi ve were climbed by the female and reached photography and an Ultra-Achromatic- the fi nal mating position (Tab. 1). Takumar 1:4.5/85 mm lens and various fi lters Experiment II (2♂♂:1♀): From the 20 (UV/IR-Cut fi lter for normal photos under a males 12 males were climbed; six males daylight lamp (OSRAM Lumilux, Cool Day- reached the fi nal mating position (Tab. 1). light L58W/865, Munich), U-fi lter and UV/ Thus, 17 males were available for colour IR-Cut fi lter under a UV-torsch (MTE U303, analysis.

Entomologie heute 30 (2018) 4 JANA BECKERT, HARTMUT GREVEN & KLAUS LUNAU

Tab. 1: Composition of the experimental groups. Left: in pairs (1♂ : 1♀); right: in groups of three (2 ♂♂ : 1♀). Bold numbers = males climbed up by a female; bold numbers underlined = males in ﬁ nal mating position. Tab. 1: Zusammensetzung der Versuchsgruppen. Links: paarweise (1♂ :1 ♀); rechts: in Dreiergrup- pen (2♂♂ : 1♀). Fette römische Zahlen = Männchen, die von einem Weibchen bestiegen wurden; unterstrichene, fette Zahlen = Männchen, die die endgültige Paarungsstellung erreicht hatten.

Tab. 2: RGB-values of 17 males and colour of the pronotal spots after the subjective impression of the observer. Tab. 2: RGB-Werte von 17 Männchen and Farbe der Pronotumﬂ ecken nach dem subjektiven Eindruck des Betrachters.

The time a female needed to climb the ﬁ rst of the load of each channel changes accord- time on the male varied between 01:30 and ing to the colour of the spots. Compared 24:02 min (see Tab. 3). to green and blue spots, red spots have relatively high “Red” values. White spots 3.2. Colour analysis reveal high values for all colours and spots with an intermediate colour (yellow, orange) In the RGB analysis “Red” had always the have low “Blue” values, but high “Red” and highest value in all males. The distribution “Green” values (Tab. 2). How females of Lucihormetica verrucosa might see the pronotal spots of their mates 5

Tab. 3: 17 males used in the choice experiments ranked after the time passed until being climbed by the female (A, B, C), and increasing blue (D), green (F) and red (H) values (see Tab. 2) each followed by the corresponding rank for the time passed until being climbed by the female (E, G, I). Statistical analysis according to Spearman’s rank correlation; p < 0.05 = signifi cant; see Tab. 2) Tab. 3: 17 Männchen aus den Wahlversuchen geordnet nach der Zeit, die bis zum Aufsteigen des Weibchens verstrichen war (A, B, C), nach zunehmenden Blau- (D), Grün- (F) und Rot-Anteilen (H) (vgl. Tab. 2) jeweils gefolgt vom zugehörigen Rang nach der Zeit, die bis zum Aufsteigen des Weibchens verstrichen war (E, G, I). (vgl. Tab. 2). Statistische Analyse mit Spearman’s Rangkorre- lationskoeffi zient; p < 0.05 = signifi kant) (vgl. Tab. 2).

There was a signifi cant negative correlation tergal glands (see GEMENO & SCHAL 2004; between the males’ attractiveness and its BELL et al. 2007). Precopulatory and copula- “Blue” value (Spearman‘s rank correlation: tory behaviour of L. verrucosa correspond to p = 0.015; r = 0.57843) and “Green” value the “type I mating behaviour”. After meeting (p = 0.01782, r = 0.56618). There are no (more or less coincidentally) and mutual signifi cant results concerning “Red” values palpating, the male presents his dorsal sur- (p = 0.33317, r = 0.25) (Tab. 3). face by rising the wings, the female climbs on his back to palpate him and to nibble 4. Discussion on the tergal glands (if present at all). Then the male backs up clasping her genitalia, the The results of the present study suggest female rotates 180o and mates reach their that females of Lucihormetica verrucosa (1) fi nal linear opposed position (see GREVEN & prefer males with red spots, and (2) recog- ZWANZIG 2013). Climbing on the male and nize the red spots by their blue and green nibbling on his back can be interpreted as components and by the fact that they do sign of interest, which, however, does not not refl ect UV. necessarily lead to copulation. In our experi- Ad 1: When taking the time a female need- ments not in all cases couples reached the ed to climb the male for the fi rst time as fi nal mating position. However, in all cases measure of attractiveness, females appeared coupling (clasping and/or fi nal position) to favour “red” males. Cockroaches have was relatively short, either interrupted by several traits that might attract the mates, the mates themselves or by the observer, i. e. short- or long-distance pheromones, e.g. when the fi nal position was reached only dominance, size and secretions of the male’s towards the end of the 30 min experimental

Entomologie heute 30 (2018) 6 JANA BECKERT, HARTMUT GREVEN & KLAUS LUNAU

Fig. 2: „Red“, „yellow“, and „white“ male of Lucihormetica verrucosa. Above: Conventional photo. Below: The same males from the “cockroach-perspective” (kindly created by MARYAM MASROURI on the basis of the UV-photo, and the 2x green components of the conventional photo). Abb. 2: „Rotes“, „gelbes“ und „weißes“ Männchen von Lucihormetica verrucosa. Oben: Normalauf- nahme. Unten: Aus „schabenspeziﬁ scher“ Sicht (freundlicherweise erstellt aus dem UV-Photo und 2x den Grünkomponenten aus der Normalaufnahme von MARYAM MASROURI). observation time. Successful copulations Ad 2. The properties of the visual system usually take more than 20 min (GREVEN in glow spot cockroaches are unknown. In & ZWANZIG 2013). With regard to the ﬁ rst other cockroach species, such as the noc- phase of courtship, i.e. the encounter of the turnal Periplaneta americana, the visual system mates and mutual antennal contact, visual is highly adapted to low light levels (e.g. cues may be involved, whereas after climbing HEIMONEN et al. 2016). This species (MOTE on the male other criteria become important & GOLDSMITH 1970; PAUL et al. 1986), Blatta for the female. orientalis (MAZOKHIN-PORSHNYAKOV & CHER- How females of Lucihormetica verrucosa might see the pronotal spots of their mates 7

KASOV 1985) and Blattella germanica (KOEHLER location, and their strong dependence on et al. 1987) possess only UV and green types the diet. This has previously led to the as- of photoreceptors. Assuming that also the sumption the spots might be important for largely nocturnal cockroaches of the genus intra- and/or intersexual selection to inform Lucihormetica (see also ZOMPRO & FRITZSCHE females about the quality of potential mates 1999; VRŠANSKÝ, et al. 2012; DITTRICH et al. and males about the quality of rivals via 2015) have only these two types of photo- their carotinoid content (discussed in detail receptors, our results suggest that the blue by GREVEN & ZWANZIG 2013). This will re- and green ranges of wavelengths provide quire that the cockroaches may differentiate signifi cant information about the colour of between “white” and “red” males, which, as the pronotal spots. The green type of photo- shown above, appears possible indirectly with receptor possesses a maximal sensitivity cockroach-subjective conspicuous spots be- around 500 nm (MOTE & GOLDSMITH 1970; ing a stain. Importance of the spots for intra- PAUL et al. 1986; MAZOKHIN-PORSHNYAKOV & and/or intersexual selection does not exclude CHERKASOV 1985; KOEHLER et al. 1987) and other functions. Although not explicitely is thus most probably sensitive to perceive mentioned in the literature, natural enemies this information. The amount of refl ection of the glow spot cockroaches are certainly in the UV range of wavelengths (BECKERT various amphibians, squamates, birds and et al. 2017) could be an additional source mammals, but it is hard to believe that these of information to distinguish between dif- spots act as an aposematic signal for these ferently coloured individuals. In addition, predators (see also VRŠANSKÝ et al. 2012). after illuminating the spots with UV-light, The assumption that glow spot cockroaches fl uorescence appears in the green range of mimic the poisonous click beetle Pyrophorus wavelengths and thus could improve the nocticula that has similar truly bioluminescent signal in the green range of wavelengths. pronotal spots (VRŠANSKÝ et al. 2012), is ill Altogether, the cockroaches may be able to founded, all the more as true biolumines- receive information about the pronotal spot cence, i.e. production and emission of light colour using their green receptors suggesting through chemical means, appears still unsub- that they distinguish differently red males stantiated in glow spot cockroaches (see the through the presence of green in the refl ec- discussion in VRŠANSKÝ et al. 2012; VRŠANSKÝ tion spectrum of the spots; red spots may be & DUŠAN 2013; MERRITT 2013; GREVEN & seen as dark gray. False-colour photography ZWANZIG 2013). However, during the day shows that the white spots are much more glow spot cockroaches might appear similar conspicuous for cockroaches than the red to predatory animals as bioluminescent click ones (Fig. 2). In our choice experiments beetles, since then the bioluminescent spots females preferred males with a lower value are not active. of green in the spots, which corresponds to the most heavily red spot colour for the hu- Literature man eye. Further analysis shows a signifi cant correlation between the amount of green BECKERT, J., GREVEN, H., & LUNAU, K. (2017): refl ection and the suspected attractiveness UV-refl ection and autofl uorescence of the of the males. pronotal spots in the glowspot cockroach Lucihormetica verrucosa (Brunner von Wat- Generally, we think that the colour of the tenwyl, 1865) (Blattodea: Blaberidae) are pronotum spots is of particular signifi cance affected by carotenoid diet. Entomologie for glow spot cockroaches despite their likely heute 29: 25-33. red-blindness. The relevance of the prono- BELL, W.J., ROTH, L.M., & NALEPA, C.A. (2007): tum spots is underlined by their exposed Cockroaches: ecology, behavior, and natural

Entomologie heute 30 (2018) 8 JANA BECKERT, HARTMUT GREVEN & KLAUS LUNAU

history. The Johns Hopkins University Press, von Blüten. Biologie in unserer Zeit 47: Baltimore. 120-127. BLOUNT, J.D., & MCGRAW, K.J. (2008): Signal MERRITT, D.J. (2013): Standards of evidence for functions of carotenoid coloration. Pp. 167- bioluminescence in cockroaches. Naturwis- 188 in: BRITTON, G., LIAANEN-JENSEN, S., senschaften 100: 697-698. PFANDER, H. (eds): Carotenoids, vol. 4: Natural MAZOKHIN-PORSHNYAKOV, G.A., & CHERKASOV, functions. Birkhäuser; Basel. A.D. (1985): Spectral sensitivity of visual BYERS, J. A. (2006): Analysis of insect and plant cells of the compound eye of Blatta orientalis. colors in digital images using Java software Neurophysiology 17: 48-51. on the internet. Annals of the Entomological MOTE, M.I., & GOLDSMITH, T.H. (1970): Spec- Society of America 99: 865-874. tral sensitivities of color receptors in the DITTRICH, H., BRENNER, M., & GREVEN, H. (2015): compound eye of the cockroach Periplaneta. Die Nutzung des Freeware-Programms Journal of Experimental Zoology 173: „Tracker“ zur Bestimmung der Aktivität von 137-146. Schaben. Entomologie heute 27: 159-170. PAUL, R., STEINER, A., & GEMPERLEIN R. (1986): FRITZSCHE, I. (2013): Krabbelnde Glühbirnen- Spectral sensitivity of Calliphora erythrocephala Leuchtschaben im Terrarium. Bugs 2: and other insect species studied with Fourier 46-49. interferometric stimulation (FIS). Journal of GEMENO, C., & SCHAL, C. (2004): Sex pheromo- Comparative Physiology A 158: 669-680. nes of cockroaches. Pp. 179-247 in: CARDÉ, VRŠANSKÝ, P., & DUŠAN, C. (2013): Luminescent R.T., & MILLAR, J.G. (eds): Advances in Insect system of Lucihormetica luckae supported by Chemical Ecology. Cambridge University ﬂ uorescence lifetime imaging. Naturwissen- Press; New York. schaften 100: 1099-1101. GREVEN, H., & ZWANZIG, N. (2013): Courtship VRŠANSKÝ, P., DUŠAN, C., FRITZSCHE, I., HAIN, mating, and organisation of the pronotum in M., & ŠEVIČIK, R. (2012): Light-mimicking the glowspot cockroach Lucihormetica verrucosa cockroaches indicate Tertiary origin of recent (Brunner von Wattenwyl, 1865) (Blattodea: terrestrial luminescence. Naturwissenschaften Blaberidae). Entomologie heute 25: 77-97. 99: 739-749. GREVEN, H., WESBUER, A., BARBIAN, A., & P. JAHNS ZOMPRO, & FRITZSCHE, I. (1999): Lucihormetica (2018): Ultrastructure and carotene content fenestra n. gen., n. sp., the ﬁ rst record of lumi- of the “glowing spots” in the cockroach nescence in an orthopteroid insect (Dictyo- Lucihormetica verrucosa (Blattodea, Blaberidae). ptera: Blaberidae: Blaberinae: Brachycolini). In preperation. Amazoniana 15: 211-219. HEATH, J.J., CIPOLLINI, D.F., & STIREMANN III, J.O. (2013): The role of carotenoids and their BSc Jana Beckert derivatives in mediating interactions between Prof. Dr. Klaus Lunau insects and their environment. Arthropod- Plant Interactions 7: 1-20. Institut für Sinnesökologie HEIMONEN, K., SALMELA, I., KONTIOKARI, P., & Department Biologie WECKSTRÖM, M. (2006): Large functional Heinrich-Heine-Universität Düsseldorf variability in cockroach photoreceptors: op- Universitätsstr. 1 timization to low light levels. The Journal of D-40225 Düsseldorf Neuroscience 26: 13454-13462. E-Mail: [email protected] KOEHLER, P.G., AGEE, H.R., LEPPLA, N.C., & PATTERSON, R.S. (1987): Spectral sensitivity Prof. Dr. Hartmut Greven and behavioural response to light quality in Department Biologie the German Cockroach (Dictyoptera: Blattel- lidae). Annals of the Entomological Society Heinrich-Heine-Universität Düsseldorf of America 80: 820-822. Universitätsstr. 1 LUNAU, K., & VERHOEVEN, C. (2017): Wie Bienen D-40225 Düsseldorf Blumen sehen – Falschfarbenaufnahmen E-Mail: [email protected]