Think and creat 3 independent questions from the reading. At least 1 of the questions must be a “higher-level” question that demonstrates in depth thinking and reasoning. A good format for these types

Think and creat 3 independent questions from the reading. At least 1 of the questions must be a “higher-level” question that demonstrates in depth thinking and reasoning. A good format for these types
Original Article Change in sex pheromone expression by nutritional shift in male cockroaches Kim Jensen, a,b,c Melanie Shearman, a James Rapkin, a Matthew R. Carey, a Clarissa M. House, a,d and John Hunt a,d aCentre for Ecology and Conservation, College of Life and Environmental Sciences, University of Exeter, Tremough Campus, Penryn TR10 9FE, UK, bDepartment of Chemistry and Bioscience, Section for Biology and Environmental Science, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark, cDepartment of Bioscience, Terrestrial Ecology, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark, and dSchool of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia Received 27 December 2016; revised 24 April 2017; editorial decision 26 July 2017; accepted 5 September 2017; Advance Access publication 12 September 2017. Environmental conditions during sexual maturation impact sexual signal expression, but little is known about how individual histories of changing environmental conditions affect the intensity of male sexual advertisement. We investigated the effects of shifting dietary nutrient composition (protein vs. carbohydrates) in male Nauphoeta cinerea cockroaches on consumption, final lipid reserves, and sex pheromone levels subsequent to completing sexual maturation on a specific diet, at high and low concentration of dietary nutrients. Consumption, lipid reserves, and sex pheromone levels were highly affected by dietary nutrient composition with higher values on car – bohydrate-biased diet, and males had significantly higher and lower levels of consumption, lipid reserves, and sex pheromones when shifted to a carbohydrate-biased and a protein-biased diet, respectively, compared with males maintained on either initial diet through – out the experiment. Males shifted to a carbohydrate-biased diet at high nutrient concentration fully recouped their sex pheromone levels, attaining levels that were not significantly lower than those in males maintained on carbohydrate-biased diet at high nutrient concentration throughout the experiment. Our study shows that male sexual display in N. cinerea is plastic and highly affected by pres – ent as well as previous dietary conditions. Signaling of adaptive quality through male sex pheromones can therefore vary dynamically within the early adult life of a male in response to the nutritional composition of food that is available to ingest. This contrasts morpho – logical sexual traits in arthropods that are affected during development and are fixed at adulthood. Key words : condition dependence, lipid reserves, Nauphoeta cinerea , nutrient balance, nutritional history, sexual signaling. INTRODUCTION The handicap theory of sexual selection predicts that male sexual traits are costly to produce and maintain and therefore serve as reli – able signals of male genetic quality ( Andersson 1994 ; Johnstone 1995b ; Hunt et  al. 2004b ; Andersson and Simmons 2006 ). According to this model, the magnitude of sexual trait expression reflects the portion of resources that the male has been able to allocate toward sexual investment, above and beyond that which is needed to maintain his basic vital requirements ( Rowe and Houle 1996 ; Tomkins et al. 2004 ). For species that have to actively search their environment for resources, these signals should express a com – bination of how good the male is at locating resources, his com – petitive abilities for accessing the resource, and how physiologically efficient he is at utilizing ingested nutrients for basic requirements thereby enabling a larger allocation to sexual display. In nature, opportunistically foraging omnivores rarely live under constant environmental conditions, yet studies investigating the effects of condition on male sexual display have all been conducted under constant conditions. Furthermore, most studies have manipulated condition at the juvenile stage and measured performance in the adults ( Rantala et  al. 2003 ; Hunt et  al. 2004a ; Ming and Lewis 2010 ; Weddle et  al. 2012 ). In contrast, the effect of condition on the plasticity of male sexual displays after reaching sexual matu – rity has been little investigated. More specifically, it has not been investigated how the history of shifting nutritional conditions on reaching adulthood may affect the condition-dependent expression of male sexual traits. The quantity of male sex pheromones has been shown to influ – ence male attractiveness to females in a range of arthropod species (Johansson and Jones 2007 ; Ruther et  al. 2009 ; Kelly et  al. 2012 ; Steiger and Stökl 2014 ; Ingleby 2015 ), and the expression of sex Address correspondence to J. Hunt. E-mail: [email protected] . Behavioral Ecology (2017), 28(6), 1393–1401. doi:10.1093/beheco/arx120 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 The Author 2017. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please e-mail: [email protected] The o cial journal of the ISBE International Society for Behavioral Ecology Behavioral Ecology Edito s choice Behavioral Ecology pheromones is found to be highly affected by nutritional conditions during development and sexual maturation (e.g., ; Rantala et  al. 2003 ; Ming and Lewis 2010 ; Weddle et  al. 2012 ; Ingleby et  al. 2013 ). For example, starved male beetles ( Tenebrio molitor ) produce significantly lower levels of sex pheromones than fed individuals (Rantala et  al. 2003 ), and male Tribolium castaneum that develop on diluted diets have lower sex pheromone levels than males develop – ing on undiluted diets which causes lower attractiveness to females (Ming and Lewis 2010 ). Furthermore, male decorated crickets (Gryllodes sigillatus ) that consume a diet of higher nutritional quality as a nymph show enhanced cuticular hydrocarbon (CHC) expres – sion as adults ( Weddle et  al. 2012 ), and in Drosophila simulans , both larval diet and temperature during sexual maturation significantly affected CHC expression in mature adults ( Ingleby et  al. 2013 ). Male lipid reserves often correlate with female preference and male mating success ( Otronen 1995 ; Cotton et  al. 2004 ; Tomkins et  al. 2004 ), and lipids as well as dietary carbohydrates are fundamen – tal components in the synthesis of sex pheromones in arthropods (Chase et  al. 1990 ; Schal et  al. 1991 ; Foster and Anderson 2015 ). However, very little is known about the exact nutrient(s) responsi – ble for the observed effects of diet on sex pheromone expression. Furthermore, the extent to which male sex pheromone expression is a plastic trait that may change according to shifting nutritional conditions after sexual maturation has not, to our knowledge, been investigated. Omnivorous animals are often found to regulate protein intake more tightly than carbohydrate and lipid intake ( Sørensen et al. 2008 ; Jensen et al. 2013 ). This includes a limited willingness to ingest protein (P) in excess of requirements due to deleterious effects of excessive P consumption. Cockroaches in particular tend not to over-ingest P relative to their requirements ( Raubenheimer and Jones 2006 ; Jensen et al. 2015 ), which is most likely linked to a limited ability to excrete nitrogenous waste products as a conse – quence of adaptation to low nitrogen availability ( Cochran 1985 ; Mullins 2015 ). In the ovoviviparous cockroach Nauphoeta cinerea , the male sex pheromone blend consists of 3 major components: 2-methylthiazolidine (2MET), 4-ethyl-2-methoxyphenol (4E2M), and 3-hydroxy-2-butanone (3H2B) ( Sréng 1990 ). Males with increased amounts of all 3 components are more attractive to females both at a distance and on contact ( Moore 1997 ; Moore and Moore 1999 ). Expression of the 3 sex pheromones is affected by temperature and food availability during sexual maturation ( Clark et al. 1997 ), and more recent experiments have shown that nutrient concentration and the specific balance of dietary P to carbohydrate (C) intake during sexual maturation has pronounced influence on lipid reserves and sex pheromone expression ( South et al. 2011 ; Bunning et al. 2016 ), as well as the attractiveness of male N. cinerea to females. In particular, each of these male traits were shown to increase with C intake but not P intake, being maximized at a P:C ratio of 1:8 ( South et al. 2011 ; Bunning et al. 2016 ). These studies, however, did not examine the effects of shifting male dietary nutri – ent composition subsequent to sexual maturation. In this study, we varied the specific nutritional history of newly eclosed adult male N.  cinerea in a factorial design of good (G; C-biased) and bad (B; P-biased) dietary nutrient concentration in a 10-day temporal sequence (G →G, B →G, G →B, or B →B) at high and low total macronutrient concentration. We measured the intake of diet (and therefore nutrients) by males, as well as the degree of lipid deposition and sex pheromone expression using gas chroma – tography-mass spectrometry (GC-MS). Based on our previous work showing that lipid deposition ( South et  al. 2011 ) and pheromone expression ( South et  al. 2011 ; Bunning et  al. 2016 ) are maximized at a high intake of C and low intake of P, we predict that both traits will be increased on a G diet and reduced on a B diet. If pheromone expression and lipid deposition are flexible and able to respond rapidly to changes in the nutritional environment, we predict that the expression of these traits will depend critically on the order that G and B diets are consumed. We predict, however, that the ability of males to recoup lipid reserves and pheromone expression (relative to males on the G →G diet) is greater on diets with high than on diets with low nutrient concentration. This will be reflected as a significant interaction between nutritional history and nutrient concentration on dietary consumption, lipid reserves, and pheromone expression. METHODS Animals and housing Individual N. cinerea were obtained from our mass culture consisting of more than 200 000 cockroaches distributed across 10 plastic con – tainers (50 cm × 35 cm × 30 cm), which are kept in an incubator at 28  ±  1  °C and a 14L:10D regime. The culture was maintained by providing a cupful of pelleted rodent chow (Rat and Mouse No.1 Maintenance, Special Diet Services, Essex, UK) containing approx – imately 12.92% digestible protein, 49.02% digestible carbohydrate, 2.47% digestible lipid, and 17.05% fiber, and 2 cotton-plugged water tubes (3-cm diameter, 15-cm long) weekly to each container. Individuals were transferred across containers at regular intervals to maintain the culture as one large, genetically mixed population. We established an isolated culture of male final instar nymphs from the mass colony and provided ad libitum rat chow and water. Males were allocated randomly to experimental treatments on their day of eclosion to adulthood (see below). During the experiment, male cockroaches were housed individually in transparent plastic con – tainers (17 cm × 12 cm × 6 cm). The experiment was performed in a constant temperature room at 28 ± 1 °C with a 14L:10D regime similar to conditions in the mass colony. Artificial diets We produced 4 artificial diets differing in quality by varying the dietary P:C ratio and total macronutrient concentration ( Table  1 ), following the protocols of Simpson and Abisgold (1985) and Dadd (1961) . These diets are identical to diets 2, 4, 22, and 24 in South et  al. (2011) and Bunning et  al. (2015 , 2016 ). Diets with a good nutritional balance contained a P:C ratio of 1:8, and diets with a bad nutritional balance contained a P:C ratio of 5:1 ( Table  1 ). Protein plus carbohydrate constituted 84% of diet dry mass at high nutrient concentration and 36% of diet dry mass at low nutrient concentration. High and low dietary macronutrient concentrations were created by adding different ratios of crystalline α-cellulose (Table 1 ), which functioned as a bulking agent. Because protein and carbohydrate contain equal amounts of metabolizable energy ( FAO 2003 ; Buchholz and Schoeller 2004 ; Simpson and Raubenheimer 2012 ), the diets were isocaloric within each macronutrient concen – tration. All diets in addition contained equal amounts of choles – terol, linoleic acid, ascorbic acid, salt mixture, and vitamin mixture (Table 1 ). Experimental design We used a 2-way factorial design to examine the effects of nutri – tional history and total nutrient concentration on diet, nutrient and 1394 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 Jensen et al. • Sex pheromone expression and nutritional history energy consumption, as well as on subsequent lipid reserves and sex pheromone expression. Within each dietary nutrient concentration (high and low), male cockroaches were allocated at random to 1 of 4 nutritional history regimes ( n = 30 males per regime, total n = 240 males). Half of the cockroaches within each level of nutri – ent concentration were provided with diet of either good (G; P:C = 1:8) or bad (B; P:C = 5:1) nutrient composition for the first 10 days posteclosion: this represents the period of sexual maturation in male N. cinerea . For each level of nutrient concentration, the diet was then shifted to the opposite diet for half of the cockroaches while the other half continued receiving the same diet for a further 10 days posteclosion. Within each total nutrient concentration, this produced the following 4 nutritional history regimes: good nutrient composition for all 20 days posteclosion (G →G), bad nutrient com – position for 10 days then good nutrient composition for 10 days posteclosion (B →G), good nutrient composition for 10 days then bad nutrient composition for 10 days posteclosion (G →B), or bad nutrient composition for all 20 days posteclosion (B →B). On day 20 posteclosion the cockroaches were killed by freezing at −80 °C. Fresh diet and water was provided in ad libitum amounts every 5  days during the experiment. Diet was provided as a powder in feeding platforms created by gluing an upturned plastic lid (1.6-cm diameter, 1.0-cm deep) to the center of an inverted petri dish lid (5.5-cm diameter, 0.5-cm deep). Water was provided in an upturned plastic lid (2.0-cm diameter, 1.0-cm deep) glued to the center of a petri dish bottom (5.4-cm diameter, 1.0-cm deep). Measuring diet, nutrient, and energy consumption The dry mass of diet in each feeding platform was weighed before and after each feeding period using an electronic balance (Ohaus Explorer Professional, model EP214C). Before weighing, diets were dried in a drying oven (Binder model FD 115, Tuttlingen, Germany) at 30  °C for 48  h. Any feces were removed with a pair of forceps before drying. Diet consumption was calculated as the difference in diet dry mass before and after feeding. P and C intake was calculated by multiplying dry mass consumption with the pro – portion of each nutrient in the ingested diet. Energy intake was calculated as the sum of energy ingested from each macronutrient group, calculated as 16.7 J per mg of P and C eaten ( FAO 2003 ; Buchholz and Schoeller 2004 ). Measuring sex pheromones To measure male sex pheromone expression, the sternum was dis – sected from each male cockroach and blotted on tissue paper to remove any lipid. The sternum was then completely submerged in 400  μL of HPLC grade dichloromethane containing an internal standard ([E,Z]-4–7-tridecadienyl acetate) at 10  ng/ μL in a 1-mL conical vial, and allowed to soak at room temperature for 2 h, after which the sternum was removed. Using an autosampler (Agilent 7683B), 2  μL of the sample extract was injected into a DB-Wax column (30 m × 0.25  mm × 0.25  μm film thickness) housed in an Agilent 7890 GC coupled with an Agilent 5975 mass spectrometer with helium as carrier gas. The inlet temperature was set at 200 °C and the injection was in pulsed split less mode. After injection, the column was held at 50  °C for 1.5  min before the temperature was raised at a rate of 10  °C/min to 250  °C, with a final hold time of 2 min. The MS transfer line was set at 240 °C. The mass spectrom – eter was operated in selected ion mode to limit the output to the target compounds (2MET, 4E2M, and 3H2B), with analysis limited to ions 45, 88, 103, 79, 107, 137, and 152. Samples were quantified against a multilevel calibration curve that we prepared using solu – tions containing the 3 target compounds at known concentrations. Analytical grade (99% pure) 3H2B was purchased from Sigma– Aldrich (St. Louis, MO), 4E2M was purchased from Pfaltz and Bauer (Waterbury, CT), and 2MT was purchased from Endeavor Specialty Chemicals (Daventry, UK). Measuring lipid reserves After removing the sternum for pheromone analysis, each cock – roach was cut along the ventral side and dried at 60  °C for 48  h, after which dry mass was weighed with an electronic balance. Lipids were extracted by placing the dry cockroach in a sealed glass vial containing 20  mL of a 2:1 solution of dichloromethane:methanol and rotating the vial at 100  rpm for 48  h on an orbital shaker (IKA-Werke, KS501 digital). Cockroaches were then removed from Table 1 Ingredient compositions of the 4 artificial diets Nutrient composition (nutrient concentration) Ingredients (per 100 g) Good (high) Bad (high) Good (low) Bad (low) Carbohydrate (g) a 74.67 14.00 32.00 6.00 Sucrose (g) 37.33 7.00 16.00 3.00 Dextrin (g) 37.33 7.00 16.00 3.00 Protein (g) b 9.33 70.00 4.00 30.00 Casein (g) 5.60 42.00 2.40 18.00 Peptone (g) 1.87 14.00 0.80 6.00 Albumin (g) 1.87 14.00 0.80 6.00 α-cellulose (g) 12.00 12.00 60.00 60.00 Cholesterol (g) 0.55 0.55 0.55 0.55 Linoleic acid (mL) 0.55 0.55 0.55 0.55 Ascorbic acid (g) 0.28 0.28 0.28 0.28 Salt mixture (g) c 2.50 2.50 2.50 2.50 Vitamin mixture (g) d 0.18 0.18 0.18 0.18 aIncludes sucrose and dextrin.bIncludes casein, peptone and albumin.cWesson’s salt mixture.dIncludes choline chloride (70.38%), meso-inositol (14.08%), nicotinic acid (5.63%), calcium pantothenate (2.82%), folic acid (1.41%), p-aminobenzoic acid (1.41%), pyridoxine (1.41%), riboflavin (1.41%), thiamine (1.41%), and biotin (0.06%). All percentages are mass based. 1395 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 Behavioral Ecology the solution and their lean dry masses were weighed after drying at 60 °C for 48 h. Lipid content was calculated by subtracting lean dry mass from total dry mass and expressed as a percentage of the initial dry mass of the cockroach (before lipid extraction) to control for variation in the body size of males. Statistical analysis The data were analyzed in a 2-factor design with nutritional his – tory regime and diet nutrient concentration as factors. Total diet consumption, P intake, C intake, and energy intake were analyzed using multivariate analysis of variance (Manova) with nutritional his – tory, diet nutrient concentration, and their interaction as fixed effects. This Manova was followed by univariate analysis of variance (Anova) to determine which trait(s) contributed to any overall multivariate effects and Fisher LSD post hoc analysis (at α  =  0.05) was used to determine which specific treatments differed significantly. Lipid reserves (expressed as a percentage of initial dry body mass) and the expression of 3H2B, 2MT, and 4E2M were analyzed using the same Manova procedure followed by post hoc analyses. Within each treatment, we determined if diet consumption differed significantly between the 2 feeding periods using a paired t-test (at α =  0.05). RESULTS Diet, nutrient, and energy consumption Nutritional history and diet nutrient concentration both signifi – cantly affected the consumption of diet, specific nutrients (P and C), and energy, and there was also a significant interaction between these main effects ( Table  2 ). On average, males consumed more diet when provided with the carbohydrate-biased diet (G) than when provided with the protein-biased diet (B) irrespective of the nutrient concentration of the diet ( Figure  1a ). Furthermore, males provided with only G diet consumed significantly more diet, whear – eas males provided only with B diet consumed significantly less diet over the experiment than males with shifting diets at both nutrient concentrations ( Figure  1a ). At both nutrient concentrations, males shifted to a G diet after restriction to a B diet significantly increased consumption in the second feeding period relative to the first, approaching but not reaching the total consumption of males pro – vided exclusively with the G diet ( Figure  1a ). Conversely, at both nutrient concentrations, males shifted to the B diet after restriction to G diet significantly reduced their diet consumption in the second feeding period and thus approached (but again did not fully reach) the low overall dietary consumption of males exclusively restricted to B diet ( Figure 1a ). Because much more G than B diet was consumed, there was a high resemblance between total dietary consumption and C intake (Figure  1b ) but no resemblance with P intake ( Figure  1c ). As the diets were isocaloric within nutrient concentrations, energy intake also resembled total nutrient consumption but with an obvious lower energy intake on the diets with low nutrient concentration (Figure  1d ). For diet consumption, C intake and energy intake, the significant interaction term between nutritional history and nutri – ent concentration occurs because the observed reduction in con – sumption across the G →G, B →G, G →B, and B →B nutritional history treatments was greater on diets with high than diets with low nutrient concentration ( Figure  1a ,b,d). In the case of P intake, the interaction between nutritional history and nutrient concentra – tion occurs because the mean intake of P in the different nutri – tional history treatments was highly affected by dietary nutrient concentration ( Figure 1c ). Lipid reserves and sex pheromone expression We found highly significant effects of nutritional history and dietary nutrient concentration, as well as the interaction between these main effects, on the amount of lipid reserves ( Figure 2a ) and on the expression of the three sex pheromones 3H2B ( Figure 2b ), 2MT ( Figure 2c ), and 4E2M ( Figure 2d ) (Table 3 ). At both nutri – ent concentrations, lipid reserves, and quantities of 3H2B, 2MT, and 4E2M were highest when males exclusively consumed the G diet and lowest when males exclusively consumed the B diet ( Figure Table 2 Manova and univariate Anova tests examining the effects of nutritional history of dietary nutrient composition (good →good, bad →good, good →bad, or bad →bad), dietary nutrient concentration (high or low), and their interaction on total diet consumption, carbohydrate (C) intake, protein (P) intake, and energy intake in the male Nauphoeta cinerea at 20 days posteclosion Model term Manova Pillai’s trace F df P Nutritional history 0.962 36.527 9696 0.0001 Nutrient concentration 0.912 795.857 3230 0.0001 History × concentration 0.770 26.691 9696 0.0001 Univariate Anova Trait F df P Nutritional history Consumption 83.345 3232 0.0001 C intake 181.633 3232 0.0001 P intake 59.995 3232 0.0001 Energy intake 74.115 3232 0.0001 Nutrient concentration Consumption 10.973 1232 0.0011 C intake 356.818 1232 0.0001 P intake 169.452 1232 0.0001 Energy intake 392.676 1232 0.0001 History × concentration Consumption 3.126 3232 0.0266 C intake 42.094 3232 0.0001 P intake 15.919 3232 0.0001 Energy intake 21.931 3232 0.0001 1396 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 Jensen et al. • Sex pheromone expression and nutritional history Figure 1 Individual consumption (mean ± SE) of (a) diet, (b) carbohydrate, (c) protein, and (d) metabolizable energy by male Nauphoeta cinerea over 20  days posteclosion, according to nutritional history regime and dietary nutrient concentration. Calculations of ingested amounts of energy are based on a general metabolizable energetic value of 16.7 J per mg for both carbohydrate and protein ( FAO 2003 ; Buchholz and Schoeller 2004 ). Gray columns show intake on dietary treatments with high total nutrient concentration (84%), and white columns show intake from dietary treatments with low total nutrient concentration (36%). Different letters indicate significant differences (Fisher LSD test, P  <  0.05). The sequence of G and B shows the sequential 10  day access to a diet with good (G; P:C  =  1:8) or a bad (B; P:C  =  5:1) nutrient composition. In (a), the second 10-day feeding period is stacked on top of the first, and error bars are for total consumption. The presence and location of the asterisk within each treatment indicates significantly larger consumption within the given feeding period than in the alternate feeding period (paired t-tests, P <  0.05). 0 (a) (b) (c) (d) 3H2B ( g) 5 10 15 20 2MT ( g) 5 10 15 20 4E2M ( g) 2 4 6 D E A A C C C B D E A A C C CD B E F A A D B D C 8 25 0 Lipid reserves (%) 0 5 10 15 20 25 D D B A C B D B 30 0 High Low Nutritional history G G G G G B G B B B B B B G B G Figure 2 Individual (mean ± SE) (a) lipid reserves as a percentage of body dry mass, and amounts of the 3 sex pheromones (b) 3-hydroxy-2-butanone (3H2B), (c) 2-methylthiazolidine (2MT), and (d) 4-ethyl-2-methoxyphenol (4E2M) in the pheromone glands of male Nauphoeta cinerea at 20  days posteclosion, according to nutritional history and dietary nutrient concentration. Gray columns represent dietary treatments with high total nutrient concentration (84%), and white columns show dietary treatments with low total nutrient concentration (36%). Different letters indicate significant differences (Fisher LSD test, P <  0.05). The sequence of G and B shows the sequential 10 day access to a diet with either a good (G; P:C  =  1:8) or a bad (B; P:C  =  5:1) nutrient composition. 1397 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 Behavioral Ecology 2a–d ). As demonstrated by the significant interaction term between nutritional history and nutrient concentration ( Table 3 ), the mag - nitude of lipid reserves and pheromone levels relative to the G →G and B →B treatments differed with nutrient concentration. At both low and high nutrient concentration, lipid reserves, and pheromone levels of males in the G →B treatment were significantly greater than in males in the B →B treatment but significantly lower than in males in the G →G treatment ( Figure 2a–d ). A similar pattern for lipid reserves and pheromone levels was shown for males in the B→G treatment when diets were of low nutrient concentration (Figure 2a–d ). However, when diets in this treatment were of high nutrient concentration, males had attained pheromone expression at the same level as those which had exclusively consumed the G diet ( Figure 2a–d ). Collectively, this demonstrates that males receiv - ing a nutritionally imbalanced diet during sexual maturation are able to partially restore their lipid reserves and fully restore phero - mone levels when shifted to a G diet, but only if the nutrient con - centration of this diet is high ( Figure 2a–d ). DISCUSSION Changes in nutritional conditions during an individual’s feed - ing history has been studied in relation to a number of key life- history traits, including growth ( Metcalfe and Monaghan 2001 ; Jespersen and Toft 2003 ; Raubenheimer and Jones 2006 ), longev - ity ( Zajitschek et  al. 2009 ), and female reproduction ( Barrett et  al. 2009 ), but nutritional changes during adult life has not before been studied in relation to male sexual signaling intensity. While it is well known that male sexual signaling is affected by nutritional conditions during development and sexual maturation ( Hunt et  al. 2004a ; South et  al. 2011 ; Weddle et  al. 2012 ), all knowledge to date is based on studies giving constant nutritional conditions dur - ing these key periods. Consequently, very little is known about how changes in the nutritional environment during adult life affect the expression of male sexual signals. This is important given that most organisms are likely to experience fluctuating nutritional conditions during their lifetime ( Simpson and Raubenheimer 2012 ), which may continuously affect the development and expression of sexual signals. We found that sex pheromone expression in adult male N. cinerea responded to shifts in dietary nutrient composition and correlated strongly with total consumption, C and energy intake, as well as body lipid reserves, all of which were inhibited when males were restricted to a protein-biased (B) diet. Thus, a male’s feeding his - tory, and the consequences for total energy intake, are essential for the maintenance of lipid reserves and sex pheromone expres - sion in male N.  cinerea . This suggests that sex pheromones in this species are energetically costly to produce and depend on a high and preferably stable intake of C and energy for maximal expres - sion. Lipid reserves and sex pheromone expression were generally affected by both the feeding period during and subsequent to sexual maturation, showing that sex pheromones in N.  cinerea are flexible and respond to the nutritional environment. When given a G diet of high nutrient concentration after a period of restriction to a nutritionally imbalanced (B) diet during sexual maturation, males were able to replenish lipid reserves and boost sex pheromone pro - duction to maximal levels. This conclusively shows that poor nutri - tional conditions during sexual maturation will not necessarily have a detrimental effect on male attractiveness in this species, as long as males are able to gain nutrients that were previously deficient. Conversely, our work also illustrates that good nutritional condi - tions during sexual maturation do not guarantee that male sexual signals will remain maximized, especially if the future diet is defi - cient in key nutrients. Our study agrees with the recent claim that exact measures of lipid reserves are often good predictors of current body condition and potential fitness ( Wilder et al. 2015 ). While the effects of nutri - ent intake on pheromone expression are well documented in male N.  cinerea (South et  al. 2011 ; Bunning et  al. 2016 ), as well as on the chemical signals of other insect species (e.g., Fedina et  al. 2012 ; Weddle et  al. 2012 ; Rapkin et  al. 2017 ), our results are novel in showing that sexual signaling in N.  cinerea is plastic and accurately reflects recent nutrient-specific foraging history. Sexual signaling intensity in male N.  cinerea correlates with the nutritional resources (C and energy) they have recently consumed and with their current body condition (lipid reserves). This contrasts the scenario associ - ated with sexually selected morphological traits in holometabolous insects, which are fixed at eclosion and therefore cannot change after reaching the adult stage (reviewed by Toubiana and Khila 2016 ). The reliance of pheromone production in N.  cinerea on the cur - rent nutritional environment suggests that chemical signaling in this species may have evolved under fluctuating nutritional conditions where it is important to adjust the cost of sexual signaling to pre - serve energy when conditions are poor and increase signaling inten - sity when conditions are good. A  similar strategy has already been documented in female N.  cinerea , which invest less in reproduction and save resources under poor nutritional conditions, and thereby increase their probability of surviving until nutritional conditions improve ( Barrett et  al. 2008 ). In contrast to males, however, this strategy in females is not determined by the diet consumed as an adult but rather is fixed by the diet consumed as a juvenile ( Barrett Table 3 Manova and univariate Anova tests examining the effects of nutritional history of dietary nutrient composition (good →good, bad →good, good →bad, or bad →bad), dietary nutrient concentration (high or low), and their interaction on lipid reserves (%) and the expression of the three sex pheromones 3-hydroxy-2-butanone (3H2B), 2-methylthiazolidine (2MT), and 4-ethyl-2-methoxyphenol (4E2M) in the male Nauphoeta cinerea at 20 days posteclosion Model term Manova Pillai’s trace F df P Nutritional history 0.911 25.172 12 693 0.0001 Nutrient concentration 0.625 95.353 4229 0.0001 History × concentration 0.298 6.364 12 693 0.0001 Trait Univariate Anova F df P Nutritional history Lipid reserves 74.721 3232 0.0001 3H2B 106.887 3232 0.0001 2MT 73.216 3232 0.0001 4E2M 155.800 3232 0.0001 Nutrient concentration Lipid reserves 130.271 1232 0.0001 3H2B 81.009 1232 0.0001 2MT 74.730 1232 0.0001 4E2M 202.413 1232 0.0001 History × concentration Lipid reserves 10.478 3232 0.0001 3H2B 10.746 3232 0.0001 2MT 3.883 3232 0.010 4E2M 4.231 3232 0.006 1398 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 Jensen et al. • Sex pheromone expression and nutritional history et  al. 2009 ). Similar albeit far more complex effects of shifting the nutritional quality of the diet between the juvenile and adult life stage have been shown for the amount of time spent calling in male black field crickets, Teleogryllus commodus (Zajitschek et al. 2009 ). The plastic effect of recent nutritional history on pheromone expression in male N.  cinerea raises the question: what information about male quality do pheromone levels signal to potential mates? It is well established that males producing high quantities of all 3 com - ponents of the sex pheromone are more attractive to females ( Moore and Moore 1999 ), and that females mating to these preferred males live longer ( Moore et al. 2003 ), produce sons that are more attractive (Moore 1990 , 1997 ), and have a shorter development time ( Moore 1994 ; but see Moore et  al. 2003 ). Our current experiment suggests that male pheromones signal the foraging success and body condition of a male and thereby his level of adaptation to the environment and his ability to pass resources to females and offspring. Clearly, more work is needed before we will fully understand how male nutrition influences female reproduction and offspring performance. The male N. cinerea did not appear to be able to build lipid reserves from ingested P, even though P contains similar amounts of metabolizable energy as C per unit dry mass ( FAO 2003 ; Buchholz and Schoeller 2004 ). This conforms with our previous work on lipid deposition in male N. cinerea (South et al. 2011 ), as well as with recent work on Blattella ger manica (Ko et al. 2016 ), and supports find - ings in Periplaneta americana which indicate that the gluconeogenic pathway is little functional in cockroaches ( Sevala and Steele 1989 ). It has been argued that cultured male and female N. cinerea , con - trary to other omnivorous insects such as crickets ( Maklakov et al. 2008 ; Harrison et al. 2014 ), do not require dietary P for the expres - sion of reproductive traits because they have full nitrogen stores from excess P consumed during earlier life stages, which they can use to resynthezise P when P is limited in the diet ( Bunning et al. 2016 ). It would therefore be interesting to deprive male N. cinerea of P during development and determine if this influences the P requirement for sex pheromone expression as adults and the ability to store energy from P as lipids while the nitrogen is deposited in uric acid stores. While our work demonstrates a correlation between nutrient intake and pheromone production in N.  cinerea , we currently do not know the mechanistic basis of this relationship. However, sex pheromone production in a range of cockroach species (e.g., Bell and Bath 1978 ; Woodhead and Stay 1989 ; Smith and Schal 1990 ; Schal et  al. 1991 ), including male N.  cinerea (Sréng et  al. 1999 ; Kou et  al. 2008 ), is stimulated by the biosynthesis of juvenile hormone, and starvation or nutrient deficiencies are known to significantly lower rates of juvenile hormone biosynthesis in a number of cock - roach species ( Weaver and Pratt 1981 ; Woodhead and Stay 1989 ; Aclé et  al. 1990 ; Chase et  al. 1990 ; Schal and Smith 1990 ; Schal et al. 1991 , 1993 ; Young et al. 1999 ). Fatty acids are the most typical pheromone precursors in cock - roaches and other insects ( Schal 1991 , 1994 ; Juárez et al. 1992 ; Gu et al. 1995 ; Tillman et al. 1999 ), and the precursors needed for sex pheromone biosynthesis could therefore be expected to be supplied from the lipid stores or directly from feeding. However, the fatty acid pathway is unlikely for the production of the 3 pheromones produced by male N. cinerea and only 1 (3H2B) of the 3 phero - mone components could in theory be synthesized from carbohy - drate through pyruvate, while the other 2 components (2MT and 4E2M) are phenolics and cannot be synthesized from lipids or car - bohydrate. Instead, these would need to be sequestered from other components, for example amino acids, as it is unlikely that N. cinerea can make phenolic rings de novo. Carbohydrate intake and lipid stores thus support the energetic requirements for pheromone syn - thesis but do not directly provide the precursors for these constitu - ents. The precursors may come from other constituents in the diet, however, maybe through symbiotic interactions with gut microor - ganisms ( Schauer et al. 2014 ; Pérez-Cobas et al. 2015 ; Tinker and Ottesen 2016 ). In conclusion, our study shows that sex pheromone expres - sion in male N.  cinerea is highly plastic, being heavily influenced by nutrient concentration and recent nutritional history. After restriction to P-biased (B) diet during sexual maturation, males were able to fully recoup lipid reserves and pheromone levels if given a C-biased diet that had a high nutrient concentration, but not when the diet was of low nutrient concentration. Our study therefore adds to the growing literature documenting the specific nutrients responsible for the condition-dependent expression of male chemical signals (e.g., South et  al. 2011 ; Fedina et  al. 2012 ; Bunning et al. 2016 ; Rapkin et al. 2017 ). More importantly, how - ever, our work demonstrates the inherent flexibility that can exist in condition dependent chemical signals due to their reliance on feeding and nutrition. Although compensatory feeding for specific nutrients and its subsequent effects on traits such as growth is well documented in animals ( Metcalfe and Monaghan 2001 ; Jespersen and Toft 2003 ; Raubenheimer and Jones 2006 ), most studies have focused on juvenile life stages. Our work shows that nutritionally determined consumption can have equally important compensa - tory effects on the expression of phenotypic traits during adult - hood and represents a potent mechanism that enables animals to counteract the effects of poor nutritional conditions experienced in the past. This is likely to have important consequences for the functioning of sexual selection in N.  cinerea . A  central assumption of handicap models of sexual selection is that only high quality males are able to afford the high costs associated with producing and/or maintaining elaborate sexual traits ( Johnstone et al. 2009 ). Consequently, it is argued that exaggerated sexual traits reliably signal male quality in female mate choice ( Johnstone 1995a ) and male–male competition ( Johnstone et  al. 2009 ). The ability of male N.  cinerea to rapidly adjust their pheromone expression with changes in the nutritional environment is likely to compromise the reliability of this sexual trait as a signal of male quality. Exactly how this will influence the outcome of sexual selection, however, is complex and theoretical models suggest that it will depend on a variety of factors, including the degree of heterogeneity that exists in the nutritional environment ( Higginson and Reader 2009 ), the extent to which males disperse between environments ( Kokko and Heubel 2008 ), and the degree to which pheromone expression is influenced by the interaction between male genotype and the nutritional environment ( Kokko and Heubel 2008 ; Higginson and Reader 2009 ). More work is clearly needed before we can assess the full impact that the plastic response of male pheromones to shifts in the nutritional environment will have on the operation of sexual selection in N. cinerea . FUNDING This study was supported by grants awarded to J.H.  from Natural Environment Research Council (NE/G00949X/1), a University Royal Society Fellowship and a Royal Society Equipment Fund grant. J.R.  was funded by NERC studentship (awarded to J.H.) and C.M.H.  was funded by a Leverhulme Trust Early Career Fellowship. 1399 Downloaded from https://academic.oup.com/beheco/article/28/6/1393/4139803 by guest on 07 January 2022 Behavioral Ecology Conflict of Interest: The authors declare no conflict of interest. Data accessibility: Analyses reported in this article can be reproduced using the data provided by Jensen et al. (2017) . Handling editor: Dan Papaj REFERENCES Aclé D, Brookes VJ, Pratt GE, Feyereisen R. 1990. 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