Density‐mediated indirect interactions alter host foraging behaviour of parasitoids without altering foraging efficiency

1. Foraging decisions of parasitoids are influenced by host density via density‐mediated indirect interactions. However, in the parasitoid's environment, non‐suitable herbivores are also present. These non‐hosts also occur in different densities, which can affect a parasitoid's foraging behaviour. 2. The influence of non‐host densities can be expressed during the first phase of the foraging process, when parasitoids use plant volatiles to locate plants infested by their host. They may also play a role during the second phase, when parasitoids use infochemicals from the host and plant to locate, recognise and accept the host. 3. By using laboratory and field setups, it was studied whether the density of non‐host herbivores influences these two phases of the foraging behaviour of the parasitoid Cotesia glomerata as well as the parasitoid's efficiency to find its host, Pieris brassicae caterpillars. 4. The findings show that a high non‐host density, regardless of the species used, negatively affected parasitoid preference for host‐infested plants, but that the behaviour on the plant and the total host‐finding efficiency of the parasitoids were not influenced by non‐host density. 5. These results are discussed in the context of density‐mediated indirect interactions.     

Relationships between parasitoid host range and host defence: a comparative study of egg encapsulation in two related parasitoid species

Abstract. Parasitoid host range may proceed from traits affecting host suitability, traits affecting parasitoid foraging behaviour, or both. We tested the hypothesis that encapsulation can be used as a reliable indicator of parasitoid host range in two closely related larval endoparasitoids of Lepidoptera. Cotesia glomerata (L.) (Hymenoptera: Braconidae) is gregarious and a generalist on several species of Pieridae, whereas C. rubecula (Marshall) is solitary and specific to Pieris rapae (L.). We determined the effects of host species ( Pieris brassicae (L.), P. napi (L.) and P. rapae ) (Lepidoptera: Pieridae) and host developmental stage (early first, second and third instar) on encapsulation of parasitoid eggs. Host species and parasitoid species, as well as the resulting interaction between these two factors had significant effects on encapsulation of Cotesia eggs. Encapsulation in Pieris hosts was much lower for C. glomerata (<34%, except for second and third instar of P. rapae ) than for C. rubecula (>32%), even when the latter was parasitizing P. rapae. Encapsulation increased with the age of the larvae, although the only significant difference was for C. glomerata. Overall, P. rapae showed a stronger encapsulation reaction than P. brassicae and P. napi. Encapsulation levels of C. glomerata corresponded well to patterns of female host species and host age preference for oviposition and parasitoid larval performance. In contrast, percentages of encapsulation of C. rubecula were not consistent with host preference and host suitability. We argue that encapsulation alone is unlikely to provide a sufficient explanation for C. glomerata and C. rubecula host range.     

Associative learning of complex odours in parasitoid host location

In this paper we address the question how hymenopteran parasitoids deal with complex odour bouquets, using Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) as a model. We examined the capacity of Microplitis croceipes to respond to individual compounds in flight chamber experiments after conditioning parasitoids with a mixture consisting of 2-octanone, methyl jasmonate and beta-caryophyllene. Parasitoids were given a choice between single compounds from the training mixture and beta-ocimene as an unrewarded alternative. When compared with control individuals lacking experience with the odour mixture, parasitoids trained to the odour blend showed an increased preference for 2-octanone and beta-caryophyllene, but not for methyl jasmonate. However, when trained with methyl jasmonate alone, parasitoids were able to respond to this compound. This demonstrates that parasitoids can learn to respond to individual compounds following experience with an odour mixture. However, for certain compounds of a mixture, learning can be blocked by other mixture components. Further experiments in which parasitoids were conditioned and challenged with two compound mixes confirmed that the olfactory background can affect recognition of individual compounds.     

Food plant and herbivore host species affect the outcome of intrinsic competition among parasitoid larvae

1. In nature, several parasitoid species often exploit the same stages of a common herbivore host species and are able to coexist despite competitive interactions amongst them. Less is known about the direct effects of resource quality on intrinsic interactions between immature parasitoid stages. The present study is based on the hypothesis that variation in the quality or type of plant resources on which the parasitoids indirectly develop may be complementary and thus facilitate niche segregation favouring different parasitoids in intrinsic competition under different dietary regimes. 2. The present study investigated whether two herbivore species, the cabbage butterflies Pieris brassicae and Pieris rapae (Pieridae), and the quality of two important food plants, Brassica oleracea and Brassica nigra (Brassicaceae), affect the outcome of intrinsic competition between their primary larval endoparasitoids, the gregarious Cotesia glomerata (Braconidae) and the solitary Hyposoter ebeninus (Ichneumonidae). 3. Hyposoter ebeninus is generally an intrinsically superior competitor over C. glomerata. However, C. glomerata survived more antagonistic encounters with H. ebeninus when both developed in P. brassicae rather than in P. rapae caterpillars, and while its host was feeding on B. nigra rather than B. oleracea. Moreover, H. ebeninus benefitted from competition by its higher survival in multiparasitised hosts. 4. These results show that both plant and herbivore species mediate the battleground on which competitive interactions between parasitoids are played out and may affect the outcomes of these interactions in ways that enable parasitoids to segregate their niches. This in turn may promote coexistence among parasitoid species that are associated with the same herbivore host.     

Effects of Pieris host species on life history parameters in a solitary specialist and gregarious generalist parasitoid (Cotesia species)

Host specificity and host selection by insect parasitoids are hypothesized to be correlated with suitability of the hosts for parasitoid development. The present study investigates the correlation between host suitability and earlier studied host-finding behaviour of two closely related braconid larval parasitoid species, the generalist Cotesia glomerata (L.) and the specialist C. rubecula (Marshall) (Hymenoptera: Braconidae). We compared the capability of both parasitoid species to parasitize and develop in three Pieris host species, i.e. P. brassicae (L.), P. rapae (L.) and P. napi (L.) (Lepidoptera: Pieridae). In laboratory experiments, we measured the effect of host species on fitness parameters such as survival, development, sex ratio and size of parasitoid progeny. The results show that C. glomerata is capable of developing in the three host species, with significant differences in parasitoid survival, clutch size and adult weight among Pieris species. The host range for development was more restricted for C. rubecula. Although C. rubecula is physiologically able to develop in P. brassicae larvae, parasitoid fitness is negatively affected by this host species, compared to its most regular host, P. rapae. A comparison of the present data on host suitability with earlier studies on host-searching behaviour suggests that the host-foraging behaviour of both parasitoid species not only leads to selection of the most suitable host species for parasitoid development, but also plays a significant role in shaping parasitoid host range.     

Powdery mildew suppresses herbivore‐induced plant volatiles and interferes with parasitoid attraction in Brassica rapa

The co‐occurrence of different antagonists on a plant can greatly affect infochemicals with ecological consequences for higher trophic levels. Here we investigated how the presence of a plant pathogen, the powdery mildew Erysiphe cruciferarum, on Brassica rapa affects (1) plant volatiles emitted in response to damage by a specialist herbivore, Pieris brassicae; (2) the attraction of the parasitic wasp Cotesia glomerata and (3) the performance of P. brassicae and C. glomerata. Plant volatiles were significantly induced by herbivory in both healthy and mildew‐infected plants, but were quantitatively 41% lower for mildew‐infected plants compared to healthy plants. Parasitoids strongly preferred Pieris‐infested plants to dually‐infested (Pieris + mildew) plants, and preferred dually infested plants over only mildew‐infected plants. The performance of P. brassicae was unaffected by powdery mildew, but C. glomerata cocoon mass was reduced when parasitized caterpillars developed on mildew‐infected plants. Thus, avoidance of mildew‐infested plants may be adaptive for C. glomerata parasitoids, whereas P. brassicae caterpillars may suffer less parasitism on mildew‐infected plants in nature. From a pest management standpoint, the concurrent presence of multiple plant antagonists can affect the efficiency of specific natural enemies, which may in turn have a negative impact on the regulation of pest populations.     

Herbivore species identity rather than diversity of the non‐host community determines foraging behaviour of the parasitoid wasp Cotesia glomerata

Extensive research has been conducted to reveal how species diversity affects ecosystem functions and services. Yet, consequences of diversity loss for ecosystems as a whole as well as for single community members are still difficult to predict. Arthropod communities typically are species‐rich, and their species interactions, such as those between herbivores and their predators or parasitoids, may be particularly sensitive to changes in community composition. Parasitoids forage for herbivorous hosts by using herbivore‐induced plant volatiles (indirect cues) and cues produced by their host (direct cues). However, in addition to hosts, non‐suitable herbivores are present in a parasitoid's environment which may complicate the foraging process for the parasitoid. Therefore, ecosystem changes in the diversity of herbivores may affect the foraging efficiency of parasitoids. The effect of herbivore diversity may be mediated by either species numbers per se, by specific species traits, or by both. To investigate how diversity and identity of non‐host herbivores influence the behaviour of parasitoids, we created environments with different levels of non‐host diversity. On individual plants in these environments, we complemented host herbivores with 1–4 non‐host herbivore species. We subsequently studied the behaviour of the gregarious endoparasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) while foraging for its gregarious host Pieris brassicae L. (Lepidoptera: Pieridae). Neither non‐host species diversity nor non‐host identity influenced the preference of the parasitoid for herbivore‐infested plants. However, after landing on the plant, non‐host species identity did affect parasitoid behaviour, whereas non‐host diversity did not. One of the non‐host species, Trichoplusia ni Hübner (Lepidoptera: Noctuidae), reduced the time the parasitoid spent on the plant as well as the number of hosts it parasitized. We conclude that non‐host herbivore species identity has a larger influence on C. glomerata foraging behaviour than non‐host species diversity. Our study shows the importance of species identity over species diversity in a multitrophic interaction of plants, herbivores, and parasitoids.     

Active defence of herbivorous hosts against parasitism: Adult parasitoid mortality risk involved in attacking a concealed stemboring host

Phytophagous insects have several defence strategies to defend themselves against attack by parasitic wasps. Larval lepidopteran hosts can defend themselves actively to prevent oviposition by the parasitoid. Among the aggressive kinds of behaviour exhibited by hosts against parasitoids are violent wriggling, biting and spitting. The behaviour of the braconid parasitoid Cotesia sesamiae attacking stemboring larvae inside their feeding tunnel in the plant stem was investigated in maize and sugarcane stem pieces and transparent artificial tunnels. Attacking a defending stemborer host inside the confined space of a tunnel was shown to be risky for the female parasitoid. A considerable proportion (25%) of female wasps were killed in their attempt to attack the spitting and biting host. No difference was found in the behaviour of C. sesamiae attacking the suitable host Sesamia calamistis or the unsuitable host Eldana saccharina. The consequences of this high mortality risk involved in each host attack is discussed in relation to the ecology of the parasitoid.     

The role of volatiles from cruciferous plants and pre-flight experience in the foraging behaviour of the specialist parasitoid Cotesia plutellae

The braconid Cotesia plutellae is an important larval parasitoid of the diamondback moth (Plutella xylostella), a major pest of crucifers in the tropics and sub-tropics. The in-flight searching behaviour of C. plutellae was investigated in a wind tunnel and the close-range attack behaviour observed in cages. The relative importance of volatile stimuli emanating from the plant-host-complex, oilseed rape (Brassica napus) – P. xylostella, in the long-range attraction of C. plutellae was investigated. Plants that were mechanically damaged, or damaged by P. xylostella larvae, were attractive to the parasitoid. Host-damaged leaves remained attractive to the parasitoid after removal of the host larvae. These results indicate that C. plutellae predominantly uses plant derived stimuli in its in-flight searching behaviour. An oviposition experience or contact with a host-damaged leaf prior to the bioassay significantly increased the response to these volatile cues. The foraging behaviour of C. plutellae is compared with other braconid larval parasitoids attacking lepidopteran hosts on crucifers.     

The combined effect of host and food availability on optimized parasitoid life‐history traits based on a three‐dimensional trade‐off surface

The reproductive success of many insects is considered to be limited by two main factors: the availability of mature eggs to lay (termed egg limitation) and the time to locate suitable hosts (termed time limitation). High host density in the environment is likely to enhance oviposition opportunities, thereby selecting for higher investment in egg supply. In contrast, a shortage of food (e.g. sugar sources) is likely to increase the risk of time limitation, thereby selecting for higher allocation to initial energy reserves. To our knowledge, the combined effect of host and food availability on these optimal life‐history allocations has never been investigated. We thus modelled their simultaneous effects on a three‐dimensional trade‐off between initial investment in energy reserves, egg number and egg size, while focusing on insect parasitoids. The model was based on Monte Carlo simulations coupled with genetic algorithms, in order to identify the optimal life‐history traits of a single simulated parasitoid female in an environment in which both hosts and food are present in varying densities. Our results reproduced the simple predictions described above. However, some novel predictions were also obtained, especially when specific interactions between the different factors were examined and their effects on the three‐dimensional life‐history surface were considered. The work sheds light on long‐lasting debates regarding the relative importance of time versus egg limitation in determining insect life‐history traits and highlights the complexity of life‐history evolution, where several environmental factors act simultaneously on multiple traits.     

Competitor‐free space mediates non‐target impact of an introduced biological control agent

Abstract 1. Enemy‐free space has been shown to mediate host shifts in herbivores, but this has not previously been documented in parasitoids. Also, natural enemies shown to maintain host shifts have always been from higher trophic levels, rather than competitors. 2. In Hawaii, an Australian parasitoid (Diachasmimorpha tryoni) of medflies that loses competition contests to a subsequently introduced Asian parasitoid (Fopius arisanus) has shifted its realised host range to attack non‐target gall flies on lantana. 3. The present study demonstrates experimentally that D. tryoni reproduction is: (i) lower on medflies in coffee when F. arisanus is present than when it is absent; (ii) higher in gall flies on lantana than on medflies in coffee, when F. arisanus is present; and (iii) higher in medflies on coffee than in gall flies on lantana, when F. arisanus is absent. This meets Berdegue et al.’s (Ecological Entomology, 21 , 203–217, 1996) three conditions to confirm the importance of enemy‐free space. 4. In the field, F. arisanus is abundant on medflies, whereas D. tryoni is rare on medflies, but is the dominant parasitoid of lantana gall flies. 5. Competitor‐free space is thus shown to be a key mechanism maintaining an apparent host shift by an introduced biocontrol agent onto a non‐target species.     

Host preferences in a phonotactic parasitoid of field crickets: the relative importance of host song characters

Abstract.  1. Predators, including insect parasitoids, often eavesdrop on prey signals, and as a result, predation can have important effects on the evolution of prey signalling behaviour.
2. The phonotactic parasitoid fly, Ormia ochracea , uses the calling songs of male field crickets to locate their field crickets hosts. In the western USA, this fly parasitises the variable field cricket, Gryllus lineaticeps . Previous work with one fly population suggested that female flies, like female field crickets, preferentially orient to male songs with higher chirp rates and longer chirp durations, although a limited range of male song types was used in this previous study. The current study, with a different fly population, used field-based, two-speaker choice tests to examine: (1) the effect of male chirp rate and chirp duration on fly attraction, using a natural range of song types; and (2) the relative importance of these song types in host selection by the flies.
3. Three lines of evidence suggested that chirp rate is more important than chirp duration in host selection. (a) The flies consistently preferred higher chirp rates but only sometimes preferred longer chirp durations. (b) The flies consistently preferred higher chirp rate/shorter chirp duration songs to lower chirp rate/longer chirp duration songs. (c) Preferences for longer chirp durations could be eliminated by increasing the amplitude of the less attractive song type, while preferences for higher chirp rates could only sometimes be eliminated by increasing the amplitude of the less attractive song type.
4. Fly predation may favour lower chirp rates and shorter chirp durations in G. lineaticeps , and may have resulted in stronger selection on chirp rate than on chirp duration.     

Spatial ecology of multiple parasitoids of a patchily‐distributed host: implications for species coexistence

1. The coexistence of multiple species sharing similar but spatially fragmented resources (e.g. parasitoids sharing a host species) may depend on their relative competitive and dispersal abilities, or on fine‐scale resource partitioning. Four generalist and one specialist parasitoid species associated with the holly leaf miner, Phytomyza ilicis, in a woodland network of 127 holly trees were investigated. 2. To understand coexistence and persistence of these potential competitors, patterns of occurrence in relation to patch size and isolation, vertical stratum within patches, and incidence and abundance of potential competitors were documented. Field experiments creating empty habitat patches suggested that dispersal rather than local demographic processes determines abundance and incidence. 3. Parasitoids showed species‐specific responses to patch properties, with the incidence of species determined mostly by patch size. Parasitism rates were less clearly related to patch characteristics, but parasitism rates for most species were lower in patches where the numerically dominant parasitoid species, Chrysocharis gemma, was present. No evidence of vertical stratification was found in species composition or abundance within patches, making it unlikely that coexistence is enhanced by fine‐scale resource division. 4. Overall, the patterns detected may be attributed to the distribution of C. gemma and differences in species' ecology other than dispersal ability. The life history of C. gemma may allow it to pre‐emptively exploit a large fraction of the available hosts, avoiding direct competition with other parasitoids. In contrast, direct competition is more likely among the pupal parasitoids Cyrtogaster vulgaris, Chrysocharis pubicornis, and Sphegigaster flavicornis which have a similar biology and phenology. For these species, coexistence may be facilitated by contrasting incidence in relation to patch size and isolation.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Reshaping our understanding of species’ roles in landscape‐scale networks

In network ecology, landscape‐scale processes are often overlooked, yet there is increasing evidence that species and interactions spill over between habitats, calling for further study of interhabitat dependencies. Here, we investigate how species connect a mosaic of habitats based on the spatial variation of their mutualistic and antagonistic interactions using two multilayer networks, combining pollination, herbivory and parasitism in the UK and New Zealand. Developing novel methods of network analysis for landscape‐scale ecological networks, we discovered that few plant and pollinator species acted as connectors or hubs, both within and among habitats, whereas herbivores and parasitoids typically have more peripheral network roles. Insect species’ roles depend on factors other than just the abundance of taxa in the lower trophic level, exemplified by larger Hymenoptera connecting networks of different habitats and insects relying on different resources across different habitats. Our findings provide a broader perspective for landscape‐scale management and ecological community conservation.     

The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.     

Adaptive foraging of leaf‐cutter ants to spatiotemporal changes in resource availability in Neotropical savannas

1. Generalist herbivores feed on a wide and diverse set of species, but fine‐scale foraging patterns may be affected by the interplay between the quality, quantity and spatial distribution of host plants. 2. The foraging patterns of a prevalent Neotropical herbivore, the leaf‐cutter ant Atta laevigata, in the Brazilian Cerrado savannas were examined in order to determine if patterns observed are in concert with central‐place foraging predictions. 3. The results showed that A. laevigata acts as a polyphagous but highly selective herbivore, with ant attacks often resulting in partial defoliation of less‐preferred species and full defoliation of preferred ones. It was found, for the first time, that there is a strong and positive relationship between the relative attack frequency on plants from preferred species and foraging distance to the nest. This suggests a balance between the quality of plant resources harvested and costs involved in their transportation. It was also observed that colonies focused their harvest on preferred species in months with low availability of young leaves. Consequently, high herbivory rate was more frequent in plants attacked far away from the nest and in dry months. 4. These assessments highlight the fact that Atta colonies may become more selective as foraging distance to the nest increases and in response to fluctuations in the availability of palatable resources throughout the year. The results also show some dissimilarities in the foraging behaviours of A. laevigata when compared with other locations, suggesting that widely distributed herbivores may modify foraging strategies across their geographic range.     

The attractiveness of different odour sources from the fruit–host complex on Leptopilina boulardi, a larval parasitoid of frugivorous Drosophila spp.

In parasitoid insects, successful offspring development depends on the female’s ability to find a suitable host. Specific recognition is often based on responses to olfactory cues, but their source and nature have rarely been determined. –This paper deals with the recognition of odours involved in host location by Leptopilina boulardi[Barbotin, Carton & Kelner-Pillault] (Hymenoptera: Eucoilidae), a larval parasitoid of Drosophila species that develops in mature fruits. The nature and origin of volatile stimuli recognized among odours of the host–fruit complex, and the effect of learning on this recognition, were investigated. Oriented responses to these odours were observed in a four-armed olfactometer and were analysed with the observer software (Noldus Information Technology). Fruit odours alone (banana and pear) were not spontaneously attractive to naive parasitoids, whereas naturally-infested bananas were highly attractive. The attraction was related to the odour that adult Drosophila left on the substrate but not to Drosophila oviposition activity or larval development. A synergism between some fruit odours (banana and pear) and the odour left by adult Drosophila on damp filter paper was observed. However, when testing a non-fruit substrate (mushroom), no synergism was observed. Thus, female L. boulardi may innately recognize host–food substrate odours associated with odours from the adult stage of their host. In addition, an oviposition experience on an infested banana allows L. boulardi females to memorise the fruit odour itself through associative learning. The adaptive significance of this process is discussed.     

The evolution of host use and unusual reproductive strategies in Achrysocharoides parasitoid wasps

We studied host selection and exploitation, two crucial aspects of parasite ecology, in Achrysocharoides parasitoid wasps, which show remarkable host specificity and unusual offspring sex allocation. We estimated a molecular phylogeny of 15 Achrysocharoides species and compared this with host (plant and insect) phylogenies. This tri-trophic phylogenetic comparison provides no evidence for cospeciation, but parasitoids do show phylogenetic conservation of the use of plant genera. Patterns of sequence divergence also suggest that the parasitoids radiated more recently (or evolved much faster) than their insect hosts. Three main categories of brood production occur in parasitoids: (1) solitary offspring, (2) mixed sex broods and (3) separate (split) sex broods. Split sex broods are very rare and virtually restricted to Achrysocharoides, while the other types occur very widely. Our phylogeny suggests that split sex broods have evolved twice and provides evidence for a transition from solitary to mixed sex broods, via split sex broods, as predicted by theory.