key: cord-0293781-2hckqh5m
authors: Zeeman, Adam N.; Smallegange, Isabel M.; Steel, Emily Burdfield; Groot, Astrid T.; Stewart, Kathryn A.
title: Toward an understanding of the chemical ecology of alternative reproductive tactics in the bulb mite (Rhizoglyphus robini)
date: 2021-07-01
journal: bioRxiv
DOI: 10.1101/2021.06.30.450527
sha: 74c1599ea1f66c6e64e4386aed17e0eee7df30ac
doc_id: 293781
cord_uid: 2hckqh5m

Background Under strong sexual selection, certain species evolve distinct intrasexual, alternative reproductive tactics (ARTs). In many cases, ARTs can be viewed as environmentally-cued threshold traits, such that ARTs coexist if their relative fitness alternates over the environmental cue gradient. Surprisingly, the chemical ecology of ARTs has been underexplored in this context. To our knowledge, no prior study has directly quantified pheromone production for ARTs in a male-polymorphic species. Here, we used the bulb mite—in which males are either armed fighters that kill conspecifics, or unarmed scramblers—as a model system to gain insight into the role of pheromones in the evolutionary maintenance of ARTs. Given that scramblers forgo investment into weaponry, we tested whether scramblers produce higher pheromone quantities than fighters, which would improve the fitness of the scrambler phenotype, e.g. through female mimicry to avoid aggression from competitors. To this end, we sampled mites from a rich and a poor nutritional environment and quantified their production of the female sex pheromone α-acaridial through gas chromatography analysis. Results We found a positive relationship between pheromone production and body size, but males exhibited a steeper slope in pheromone production with increasing size than females. Females exhibited a higher average pheromone production than males. We found no significant difference in slope of pheromone production over body size between fighters and scramblers. However, scramblers reached larger body sizes and higher pheromone production than fighters, providing some evidence for a potential female mimic strategy adopted by large scramblers. Pheromone production was significantly higher in mites from the rich nutritional environment than the poor environment. Conclusion Further elucidation of pheromone functionality in bulb mites, and additional inter-and intrasexual comparisons of pheromone profiles are needed to determine if the observed intersexual and intrasexual differences in pheromone production are adaptive, if they are a by-product of allometric scaling, or diet-mediated pheromone production under weak selection. We argue chemical ecology offers a novel perspective for research on ARTs and other complex life-history traits.

The model selection procedure revealed that the effects of morph, diet and idiosoma length on 143 log(α-acaridial production) were best described by model 7 (see Table 1 ), in which the 144 relationship between idiosoma length and log(α-acaridial production) differed significantly 145 between the sexes (idiosoma length × morph; F1,50 = 15.12, p < 0.001), with males showing a 146 bigger increase in log(α-acaridial production) with increasing idiosoma length than females 147 ( Figure 3a ) (note that in model 7, fighters and scramblers are merged into one factor level 148 'males'). Additionally, females produced on average more α-acaridial than males (F1,50 = 14.33, p 149 < 0.001). Also, log(α-acaridial production) was significantly higher on the rich diet than on the 150 poor diet (rich 2.60 ± 0.66 ng, n = 29 pools; poor 1.45 ± 0.76 ng, n = 26 pools; F1,50 = 9.65, p < 151 0.01; Figure 4 ).

Inspection of the second-best fitting model (Table 1 , model 4), in which fighters and 153 scramblers were included as separate factor levels in the factor morph, revealed, like in the best-154 fitting model, that males of both morphs showed a bigger increase in log(α-acaridial production) 155 with increasing size than females (idiosoma length × morph; F2,48 = 8.34, p < 0.001) (Figure 3b ). 156 However, inspection of this second-best fitting model also shows that the highest log(α-acaridial DISCUSSION 163 In our study, we aimed to gain insight into the role of pheromones in the evolution and 164 maintenance of ARTs by assessing α-acaridial production in the male-dimorphic bulb mite under 165 two nutritional regimes. We tested the hypothesis that scramblers, given that they forgo investment into weaponized legs, can produce higher quantities of α-acaridial than fighters, 167 which would improve the reproductive success of the scrambler phenotype, e.g., through female 168 chemical mimicry. We found that α-acaridial production was positively correlated with body size, 169 and the slope of α-acaridial production over body size was steeper in males than females. In 170 addition, α-acaridial production was influenced by the nutritional environment, as mites on the 171 rich diet produced more pheromone than those on the poor diet. On average, females produced 172 more α-acaridial than males. Differences between male ARTs were not incorporated in the linear 173 model that best described the data (although they were incorporated into the second-best model), 174 indicating that there is no significant difference in slope of α-acaridial production over body size 175 between the male morphs. Nevertheless, scramblers reached larger maximum body sizes and 176 higher maximum α-acaridial production compared to fighters.

Intersexual and intrasexual differences in pheromone production 179 We found that females produced significantly more α-acaridial than males, corroborating the 180 intersexual differences in α-acaridial production described by Mizoguchi et al. [55] . Importantly, 181 the slope of pheromone production over body size was steeper in males than females, indicating 182 that intersexual differences in pheromone production are not due solely to size differences 183 between the sexes. Potentially, males may disproportionately benefit from an increased α-184 acaridial production with increased size. If, for example, pheromone production acts as an honest 185 indication of quality in bulb mites, sexual selection-which is particularly strong in species with 186 male ARTs [2]-may drive well-conditioned males to produce as much pheromone as they can, 187 without incurring high viability costs. Alternatively, the steeper slope of pheromone production 188 over body size for males may result from differences in chemical ecology between large and 189 small males. The largest males, which in this study are represented mostly by scramblers, produced some of the highest pheromone quantities in the dataset, while many of the smallest 191 males in this study produced very low quantities. This provides some evidence for the 192 hypothesized female mimicry strategy adopted by large scramblers. These large, well-193 conditioned scramblers may be the only males capable of producing enough pheromones to 194 mimic female pheromone profiles, while also reaching body sizes comparable to females. As 195 such, these scramblers may disproportionately benefit from producing high pheromone quantities 196 compared to smaller males. It should be emphasized however, that this study does not provide 197 evidence that all scramblers are female mimics, as the model that best describes the data did not 198 differentiate between male ARTs. Nevertheless, the results of this study do warrant further 199 exploration of a female mimic or "sneaking" strategy in bulb mites, particularly because 'mega-200 scramblers'-which sometimes elicit mating behavior from other males-are suggested to be a 201 third ART [47] . Indeed, these mega-scramblers may be the result of sexual selection driving 202 larger scramblers to chemically and physically resemble females. Intriguingly, some of the 203 largest scramblers in this study, which produced higher pheromone quantities than most females 204 in the dataset, exceeded a body size breakpoint for mega-scramblers calculated by Stewart et al. 205 [47] , implying these individuals may in fact represent the mega-scrambler trimorphism.

Role of nutritional environment and body size in pheromone production 208 The observed effects of body size and diet both suggest that pheromone production is linked to 209 nutritional uptake-particularly because body size was also dependent on the nutritional 210 environment. The positive correlation between body size and pheromone production may stem 211 from covariation of these variables with diet quality and uptake. As such, individuals that can 212 consume more high-quality food subsequently grow larger [17] , while simultaneously 213 maintaining a good enough condition to produce high pheromone quantities. In this context, pheromone production could be a form of honest signaling of individual quality [56] . 215 Alternatively, larger individuals may simply produce more pheromone because they have bigger 216 or better developed pheromone glands, and as such, the observed patterns in pheromone 217 production may be non-adaptive. Indeed, there is some support for allometric scaling of the 218 production of defensive chemicals in astigmatic mites, as a study on Archegozetes longisetosus 219 showed that the quantity of defensive chemicals from opisthonotal glands scales allometrically 220 with body mass during ontogeny [57] . However, it is not known if this allometric relationship To better understand the ecological significance of divergent pheromone profiles between sexes 237 and (potentially) male ARTs in bulb mites, it is imperative that pheromone functionality is further elucidated in this species. So far, functionality has only been described for two bulb mite 239 exudates; α-acaridial as a putative female sex pheromone [55] and neryl formate as an alarm 240 pheromone [64] . As a putative female sex pheromone, α-acaridial was a good candidate to 241 investigate the role of pheromones in the maintenance of bulb mite ARTs. However, evidence for 242 the sex pheromone activity of α-acaridial is limited to a single study, where the compound was 243 found to be present in the fractions of female hexane extracts that triggered mounting behavior in 244 males [55] . Synthetic α-acaridial was also shown to elicit mounting behavior at a dose of 10 ng.

However, neither of these findings prove that α-acaridial is the only female sex pheromone in 246 bulb mites. Indeed, it is possible that various compounds function synergistically (with or without also 27], and should always be considered when assessing the effects of individual compounds.

Other bulb mite exudates with potential relevance to the chemical ecology of this species 255 include neral [64] , several hydrocarbons [69) ] robinal, perillene and isopiperitenone [70] . Several 256 of these compounds have also been found in other astigmatic mites [71] , but their functionality is 257 mostly unknown in these species.

The lack of knowledge on bulb mite chemical ecology means that there are many avenues male ARTs that lack anal glands and thus a putative sex pheromone [73] . In the black goby, in 284 which males are large "parentals", small sneakers or an intermediate phenotype [74] , males 285 produce a sex pheromone that triggers aggression in other males [75] . However, parental males were found to react aggressively to the pheromone-containing ejaculate of other parentals but not stabilizing selection [77] [78] [79] [80] . Therefore, the evolutionary relationship between ARTs and 300 divergent pheromone profiles may be reversed, such that the evolution of ARTs facilitates the 301 evolution of divergent pheromone profiles. By definition, ARTs adopt different strategies to 302 improve their reproductive output, and therefore they face different selection pressures [2] . For 303 example, large males that compete for females directly will likely be favored by selection to 304 develop traits that improve their ability to fend off competitors, while smaller males that adopt 305 sneaking tactics may well be favored to develop traits that make them inconspicuous towards 306 other males. These divergent selective pressures may decouple male (sex) pheromone profiles in 307 the population from stabilizing selection, or rather, stabilizing or directional selection may now 308 occur more or less independently for the pheromone profiles of both ARTs, leading to disruptive 309 selection on the population level. There is also emerging evidence that variation in sex pheromone profiles can be maintained by balancing selection [81, 82] , e.g., through heterozygote 311 advantage [81] . Thus, within-population variation in pheromone profiles may arise and be We found a positive relationship between pheromone (α-acaridial) production and body size in 318 bulb mites, but importantly, males demonstrated a steeper slope in pheromone production with 319 increasing size than females. We found no significant difference in slope of pheromone 320 production over body size between fighters and scramblers, but scramblers reached larger 321 maximum body sizes and thus had higher maximum pheromone production compared to fighters.

The results of this study also indicate that diet quality influences pheromone production in bulb To our knowledge, this is the first study to directly quantify the production of a 334 pheromone for two ARTs in a male polymorphic species. Yet, intrasexual differences in 335 pheromone production in male-polymorphic species offer promising research avenues in the 336 context of crossing fitness functions that underlie the maintenance of these polymorphisms. 

The bulb mite 343 The blind bulb mite (Rhizoglyphus robini), a common agricultural pest that feeds on various 344 crops [83] , is an excellent model system for studying the expression and maintenance of ARTs.

In addition to its short generation time and high reproductive output, this microscopic mite can 346 easily be reared in the laboratory under various conditions [53] . After hatching, bulb mites 347 undergo four or five developmental stages: larva, protonymph, deutonymph (a facultative 348 dispersal stage that occurs under adverse conditions, such as food or water scarcity), tritonymph 349 and adult [84] . Transitions between these stages occur in the form of a quiescent molting stage. 

After that, they were moved to a location without access to climate chambers and kept at room 359 temperature. The experiments were conducted at room temperature as well. Mites were kept in 360 sealed but ventilated plastic containers (50 mm high, 85 mm in diameter) that contained a layer of 361 plaster of Paris (~15 mm thick) that was nearly saturated with water. The stock cultures were 362 either always given ad lib access to dried yeast granules (Bruggeman instant yeast), or ad lib 363 access to grains of rolled oats. Yeast and oats are of high and low nutritional quality, respectively, 364 due to their respective high and low protein content [50] . Therefore, these resources will further 365 be referred to as "rich" (yeast) and "poor" (oats) diets. To reduce inbreeding, stock populations 366 fed on the same diet were intermixed periodically, effectively creating multiple meta-populations.

Additional food and water were provided to each stock container once or twice per week (in a 368 manner similar to [44] ). The observed heterozygosity (averaged across sex and ART) of the stock Body size measurements 395 The idiosoma length (the length of the body without mouthparts; Figure 1D ) of the collected 396 mites was measured as a proxy for body size [43, 44] . First, the mites were photographed using a 397 ZEISS Axiocam 105 color camera at 0.63-5 × magnification that was connected to a Zeiss Stemi (Table 1) [85] . This procedure was repeated until the model only contained significant terms 448 (P < 0.05). It turned out that this minimal model contained the fixed factor morph, which has 449 three levels (female, scrambler, fighter). To assess which of the different levels of the factor and scramblers do not significantly differ in log(α-acaridial production). We confirmed that the 455 assumption of a Normal error distribution was justified by visual inspection of histograms of 456 model residuals and normal quantile-quantile plots, and confirmed that the assumption of 457 homogeneity of residuals was justified using residuals-versus-fits plots. All statistical analyses 458 were conducted in RStudio [86] . To ensure quantifiable amounts of pheromone could be obtained from bulb mite hexane extracts, 570 pilot extractions were performed. Individual mites and groups of mites, ranging from two to ten 571 individuals, were submerged in various amounts of hexane for different durations (Table S1 ). All 572 mites used in these extractions were randomly sampled from the stock populations, following the 573 procedure described in the Methods. The extracts were analyzed through gas chromatography 574 (GC), also following the procedure described in the Methods. Pilot extractions were deemed 575 successful when clear, quantifiable pheromone peaks were seen in the resulting chromatograms.

The results indicated that at least two females or ten males (mostly performed using fighters) 577 were required to consistently obtain measurable amounts of pheromone from a single extract.

Two additional pilot extractions were performed to check for potential contamination of yeast 579 granules and oat grains (Table S1 , bottom rows). This was done because food particles from the 580 plastic tubes that housed the mites were sometimes accidentally submerged in the hexane along 

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