Clonal mixing in the soldier-producing aphid Pemphigus spyrothecae (Hemiptera: Aphididae)

Illuminating the genetic relationships within soldier-producing aphid colonies is an essential element of any attempt to explain the evolution of the altruistic soldier caste. Pemphigus spyrothecae is a soldier-producing aphid that induces galls on the leaf petioles of its host (trees of the genus Populus). At least a quarter of the aphids within the clonally produced gall population are morphologically and behaviourally distinct first-instar soldiers that defend the gall population from predation. Using field trapping and microsatellites, we investigated the degree of clonal mixing within natural gall populations. Field trapping in the UK showed that all the migrants of P. spyrothecae and of two other Pemphigus species were wingless first-instar soldiers. The average degree of mixing estimated from trapping P. spyrothecae migrants was 0.68% (range = 0-15%). Microsatellite genotyping of 277 aphids from 13 galls collected in Italy revealed an average mixing level of 10.4% (range = 0-59%). Six galls contained more than one clone (range = 2-5 clones). Non-kin aphids were not restricted to the soldier caste but were evenly distributed across instars. An additional gall, from which 527 occupants were genotyped, contained 12 non-kin aphids distributed among nine clones, showing that clonal diversity can be high even when mixing is very low. These observations suggest that although soldiers migrate regularly and can moult and reproduce within foreign galls, clonal mixing in this species is generally low and is unlikely to provide a barrier to the evolution of investment by the aphid clones in an altruistic soldier caste.     

Defensive Behavior in Primary- and Secondary-Host Generations of the Soldier-Producing Aphid, Pemphigus bursarius (Hemiptera: Aphididae)

The genus Pemphigus comprises several species that produce soldiers (defensive morphs) in galls on the primary host. At the moment, it is unclear if host-alternating species also produce defenders on their secondary host. We therefore examined how P. bursarius morphs of the secondary host generations respond to predators to test whether they show defensive behavior. We further examined how this response compares with the antipredator behavior of soldiers in P. bursarius and P. spyrothecae occurring on the primary host. We performed two manipulative experiments using two predatory species to quantify the behavior of the different morphs in response to predators. In both experiments, secondary host morphs of P. bursarius showed no attacking behavior and antipredator behavior in these morphs was limited to escaping natural enemies by walking away. In contrast, the first instars of the primary host generations in both Pemphigus species showed attacking behavior and were capable of killing the predators.     

Soldiers of a European gall aphid,Pemphigus spyrotecae (Homoptera: Aphidoidea): Why do they molt?

A non-migratory aphid,Pemphigus spyrothecae, produces 1 st-instar larvae of 2 types in the gall: thick-legged ones and normal-legged ones. It was found that the thick-legged larvae play a defensive role, hence they may be called soldiers. Unlike the soldiers of other species hitherto reported, at least some soldiers of this species molt and become adults. Their incomplete sterility is explained historically: 1) The migratory ancestor ofP. spyrothecae once produced monomorphic 1st-instar larvae attacking predators in the gall. 2) It acquired a non-migratory life cycle by the larviposition of emigrants in the gall. 3) As a result, 1 st-instar larvae of another morph, which had lived on the secondary host without attacking predators, joined the gall inhabitants. This hypothesis is supported strongly by the fact that the normal-legged 1 st-instar larvae very much resemble the 1 st-instar larvae of migratoryPemphigus species produced on the secondary hosts.     

Maintenance of soldier-producing aphids on an artificial diet

In our efforts to understand the biology of soldier-producing aphids, we attempted to maintain them in the laboratory using a chemically defined artificial diet. The ability of 16 species from the subfamilies Eriosomatinae and Hormaphidinae, most of which are soldier-producing species, to survive on the artificial diet was examined. Some species neither fed nor grew on the diet, whereas other species accepted the diet, grew to some extent, and managed to produce a small number of short-lived offspring. Although they performed poorly on the diet in general, aphid performance was correlated with the stage in the life cycle and the developmental stadium in that aphids of the gall generation tended to accept the diet and survive on it, whereas aphids of the non-gall generation did not. Also, old insects tended to perform better on the diet than young nymphs. Notably, only one species, Tuberaphis styraci, a gall-forming aphid that produces 2nd instar sterile soldier, showed good performance on the diet. Insects collected from galls (generation G0) survived on the diet, grew well, and produced many progeny. Three successive generations (G1, G2 and G3) were produced on the diet. Developmental period, adult body size, and age of first reproduction were almost constant through G0, G1 and G2 whereas fecundity, adult longevity and daily offspring production declined as the generations proceeded. These results are comparable to previous studies in which pest aphids have been maintained on similar artificial diets for several generations. Therefore, it is suggested that the artificial-diet rearing system will provide a useful tool to investigate various biological aspects of the soldier-producing eusocial aphid, T. styraci.     

Variation in propensity to defend by reproductive gall morphs in two species of gall-forming thrips

Summary.Six species of Australian phleaothripine gall forming thrips on Acacia have two morphs. One morph, referred to as a soldier, has reduced antennae and wings and greatly enlarged fore femora, which are thought to be adaptations for gall defence. For most species, female soldier morphs have reduced reproductive output relative to dispersing females and these species have been regarded as eusocial. We examine defensive behaviour of soldier morphs using in situ presentation of gall invading kleptoparasites. We show that the gall-morph of Oncothrips tepperi attack kleptoparasites more often (N=87 interactions, n = 11 attacks) than does the gall-morph of Oncothrips morrisi (N = 231, n = 1). This difference in proclivity for defence is coincident with a lower reproductive output by the gall-morph in O. tepperi compared to O. morrisi. Our results also show an almost complete absence of attack behaviour by the gal l-morph of O. morrisi. Lastly, we show that there is no difference in proclivity for defence between soldier and foundress morphs of O. tepperi. These observations taken together call into question the suitability of the term 'soldier' as applied to the gall-morph of the gall-forming thrips of Australia.     

Fortress repair in the social aphid species Pemphigus spyrothecae

Nest repair is a vital element in the cooperative brood care that is a distinctive feature of eusocial animals. We investigated the repair role of individuals of the social aphid Pemphigus spyrothecae by prematurely opening their gall by cutting a hole of the same size and position as a natural opening. This resulted in increased mortality from predation. No subsequent responsive shift towards increased investment in soldiers was detected. However, repair of the holes by compensatory regrowth from undamaged adjoining areas of the gall was observed in 97.5% of the experimental galls that were protected from predation. Subsequent experiments confirmed that the aphids were responsible for this repair, with soldiers likely to be the major agents. Active maintenance of the integrity of the gall represents another altruistic task for which soldiers are predominantly responsible and indicates that social complexity in the order Hemiptera is greater than previously assumed.     

Reproductive schedule and factors affecting soldier production in the eusocial bamboo aphid Pseudoregma bambucicola (Homoptera, Aphididae)

Summary: The reproductive characteristics of the soldier-producing aphid Pseudoregma bambucicola were studied in Kagoshima, Southern Japan, to know the factors affecting soldier production of eusocial aphids. The soldier proportion in aphid colonies was highest from October to November. In some large colonies, soldiers were observed in all seasons except in July when colony size was relatively small. Multiple regression analysis showed that the colony size was a principal factor affecting soldier proportion throughout a year. Other social or environmental factors such as aphid composition, host plant conditions and predator abundance were not always significant. Rearing experiments revealed that large colonies (̿,000 individuals) produced soldiers in almost all seasons while small colonies (<1,000) never produced any soldiers. The caste-production schedule of adult females was examined in the field. When solitary females produced both castes, they usually produced normal nymphs first and then soldiers. Females from large colonies tended to produce more soldiers in the earlier period of their lifetime, whereas females from newly established small colonies produced no or only a few soldiers at later times. The average number of soldiers and normal nymphs produced consecutively by a single female was >10 and >20, respectively. Because they have a small number of ovarioles (<15 on average), females should alter caste production within the same ovarioles according to changes in environmental conditions. Artificial removal or introduction of predators and reduction of colony size did not affect soldier production over two successive generations, revealing maternal effects on soldier production. Females cannot shift caste production quickly in response to changes in predator abundance and colony size. This is probably due to early developmental determination of castes within the mother's body.     

Lack of kin discrimination in the eusocial aphid pseudoregmabambucicola (Homoptera: Aphididae)

The significance of kin discrimination for the evolution of sociality is theoretically important. The behavioural response ofPseudoregma bambucicola soldiers to aphids from other colonies was studied to examine the possibility of context-specific kin discrimination. When non-kin aphids were artificially introduced into experimental colonies, they were never killed or removed by soldiers. Field experiments revealed that the soldiers frequently attacked distantly related species such asAstegopteryx bambucifoliae andCeratoglyphina styracicola, but rarely conspecific aphids or congeneric species such asP. koshunensis andP. alexanderi. Another experiment showed that the soldiers showed altruistic defence even when coexisting with non-kin reproductives. These results agree with and extend the results from previous studies of kin discrimination in social aphids. The absence of kin discrimination suggests that some other factors besides higher genetic relatedness may play a prominent role in the evolutionary maintenance of soldier behaviour in aphids.     

Host behaviour and its influence on foraging and acceptance by the solitary parasitoid wasp,Venturia canescens (Hym: Ichneumonidae)

Behavioural interactions between the solitary koinobiont parasitoid,Venturia canescens, and two of its hosts,Plodia interpunctella andCorcyra cephalonica, were investigated. The response of both hosts to simulated antennation using a two-haired brush was examined over instars 3 (L3) to 5 (L5). YoungP. interpunctella larvae predominantly adopted escape tactics (writhe, trash) whereas L5P. interpunctella usually froze after the stimulus was applied. L3C. cephalonica larvae were more aggressive (headrear, flick) thanP. interpunctella in response to the application of the stimulus, but olderC. cephalonica responded less aggressively than in earlier instars. AlthoughV. canescens readily jabbed its ovipositor at both hosts after antennation,P. interpunctella was considerably more susceptible to parasitoid attack thanC. cephalonica, irrespective of size in the final (L5) instar.C. cephalonica, the larger, more aggressive host, actively resisted parasitism whereasP. interpunctella responded much more passively after parasitoid contact. Parasitoids examined and jabbed their ovipositors at dead hosts, but this behaviour was not sustained, implying that host movement stimulates parasitoid attack. On patches containingV. canescens, L5C. cephalonica andP. interpunctella, mostP. interpunctella larvae responded by freezing after parasitoid contact.P. interpunctella that froze usually avoided parasitism, whereas larvae that attempted to escape by crawling were pursued with vigour byV. canescens and usually parasitized. Irrespective of behaviour after parasitoid contact,C. cephalonia displayed more aggressive behaviour and had much greater success in warding off parasitoid attack. Host acceptance byV. canescens is clearly affected by the size and species of the host it attacks. The influence of host defensive behaviour is discussed in relation to the evolution of parasitoid counter-defences and oviposition strategies.     

Phylogeny of Rhus gall aphids (Hemiptera : Pemphigidae) based on combined molecular analysis of nuclear EF1α and mitochondrial COII genes

Rhus gall aphids (Fordinae : Melaphidini) have a disjunct distribution in East Asia and North America and have specific host plant relationships. Some of them are of economic importance and all species form sealed galls which show great variation in shape, size, structure, and galling‐site. We present a phylogeny incorporating ten species and four subspecies of Rhus gall aphids based on 1694 base pairs of nuclear elongation factor‐1α (EF1α) and mitochondrial cytochrome oxidase subunit II (COII) DNA sequence data. The results suggest that Melaphidini is monophyletic and at the genus level, Schlechtendalia, Nurudea, and Floraphis were each monophyletic. Kaburagia and Meitanaphis were not monophyletic and therefore inconsistent with the current classification. The North American sumac gall aphid, Melaphis rhois, was most closely related to the East Asian Floraphis species, although this was poorly supported. The conservation of gall morphology with respect to aphid phylogeny rather than their host plants suggests that gall morphology is largely determined by the aphids. While there is no evidence of strict co‐speciation between the aphids and their primary host plants, switching between recently diverged host plants may be involved in the speciation process in Melaphidini.     

The influence of species identity and herbivore feeding mode on top-down and bottom-up effects in a salt marsh system

In this study we investigated the potential importance of species identity and herbivore feeding mode in determining the strengths of top-down and bottom-up effects on phytophagous insect densities. In 1998, we conducted two factorial field experiments in which we manipulated host plant quality and intensity of parasitoid attack on three salt marsh herbivores, the planthoppers Prokelisia marginata and Pissonotus quadripustulatus (Homoptera: Delphacidae), which feed only on Spartina alterniflora and Borrichia frutescens, respectively, and the gall fly Asphondylia borrichiae (Diptera: Cecidomyiidae), which feeds only on B. frutescens. We increased plant quality through addition of nitrogen fertilizer, and decreased parasitism by trapping hymenopteran parasitoids continuously throughout the study. Herbivore densities were censused biweekly. Increasing plant quality through fertilization increased the density of all three herbivores within 2 weeks of treatment application, and higher densities were maintained for the duration of the study. Reduction of top-down pressure had no effect on either planthopper species, possibly because of compensatory mortality affecting the two species. In contrast, reduction of parasitism significantly increased the density of A. borrichiae galls, perhaps because development within gall tissue reduces the sources of compensatory mortality affecting this species. The results of this study show that the bottom-up effects of plant quality were strong and consistent for all three species, but the strength of top-down effects differed between the two feeding guilds. Thus, even for herbivores feeding on the same host plant, conclusions drawn regarding the relative importance of top-down and bottom-up effects may vary depending upon the feeding mode of the herbivore.     

Insects as vectors of plant pathogens: mutualistic and antagonistic interactions

Interactions between plants and their herbivores and pathogens are mostly analysed separately, thereby neglecting mutualistic or antagonistic interactions between these antagonists and possible joint effects on the host. We studied interactions between the weed Cirsium arvense, the rust fungus Puccinia punctiformis and three herbivorous insects, the aphids Aphis fabae ssp. cirsiiacanthoidis and Uroleucon cirsii, and the beetle Cassida rubiginosa. All three insect species mechanically transported spore material and significantly increased rates of P. punctiformis infection in healthy thistles. The interaction between C. rubiginosa and the fungus was antagonistic. Although C. rubiginosa transferred spores, biomass of adults was significantly reduced, development of adults tended to be prolonged and mortality increased when feeding on plants infected with P. punctiformis. In contrast, the relationship between the aphid U. cirsii and P. punctiformis was mutualistic: U. cirsii profited by fungal infection and formed significantly larger colonies on fungus-infected plants. Although the differences in insect performance suggest that aphids may be better vectors than the beetle, infection rates were similar. This is the first study to demonstrate that the relationship between herbivores, which increase the dispersal of a pathogen, and the pathogen itself can be mutualistic or antagonistic, depending on the species.     

Social parasitism and behavioral interactions between two gall-forming social aphids

We investigated ecological and behavioral aspects of the interactions between two social aphids, Pseudoregma bambucicola and Astegopteryx bambucifoliae (Hormaphidinae, Cerataphidini), both of which produce second-instar, sterile soldiers in galls formed on Styrax suberifolius, in Taiwan. By censusing their galls, either species was found to invade galls of the other species. Twenty-eight (58%) out of 48 A. bambucifoliae galls contained P. bambucicola, while four (6%) of 69 P. bambucicola galls contained A. bambucifoliae. Furthermore, P. bambucicola behaved like social parasites in galls of A. bambucifoliae. Colonies of P. bambucicola produced much fewer or even no soldiers compared with those in natal galls. Our experiments also revealed that individual aphids of P. bambucicola more successfully intruded into galls of A. bambucifoliae than into conspecific galls, and that guarding soldiers of P. bambucicola effectively prevented aphids from invading their galls and permitted only conspecific soldiers to join their colonies. These differences in behavior provide good explanations for the differences in the frequency of invaded galls found between the two species. Received 4 September 2007; revised 7 January 2008; accepted 17 January 2008.     

Evolutionary relationships of Pemphigus and allied genera (Hemiptera: Aphididae: Eriosomatinae) and their primary endosymbiont,Buchnera aphidicola

Aphids harbor primary endosymbionts, Buchnera aphidicola, in specialized cells within their body cavities. Aphids and Buchnera have strict mutualistic relationships in nutrition exchange. This ancient association has received much attention from researchers who are interested in endosymbiotic evolution. Previous studies have found parallel phylogenetic relationships between non‐galling aphids and Buchnera at lower taxonomic levels (genus, species). To understand whether relatively isolated habitats such as galls have effect on the parallel relationships between aphids and Buchnera, the present paper investigated the phylogenetic relationships of gall aphids from Pemphigus and allied genera, which induce pseudo‐galls or galls on Populus spp. (poplar) and Buchnera. The molecular phylogenies inferred from three aphid genes (COI, COII and EF‐1α) and two Buchnera genes (gnd, 16S rRNA gene) indicated significant congruence between aphids and Buchnera at generic as well as interspecific levels. Interestingly, both aphid and Buchnera phylogenies supported three main clades corresponding to the galling locations of aphids, namely leaf, the joint of leaf blade and petiole, and branch of the host plant. The results suggest phylogenetic conservatism of gall characters, which indicates gall characters are more strongly affected by aphid phylogeny, rather than host plants.     

Caste allometries in the soldier-producing aphidPseudoregma alexanderi (Hormaphididae: Aphidoidea)

Summary Colonies of the aphidPseudoregma alexanderi produce morphologically-specialized first-instar larvae, termed soldiers, that defend the colony from predators. The environmental cues and physiological mechanisms governing soldier production are currently unknown. Here we present a morphometric study of soldiers and normal first-instar larvae ofP. alexanderi. Several morphological features (fore-leg length and width, hind-leg length, and horn length) plotted against body length display relationship that are similar to a sigmoidal curve. We found further support for an earlier finding that soldiers fall into two size categories, majors and minors, although both types of soldiers appear to follow the same allometry. The patterns of allometry in the soldier-producing aphids are very different from those found in other social insects and do not easily fit into the traditional categorization of allometries. We present two simple alternative models of soldier development as a framework for guiding future studies of the mechanisms of soldier production.     

VARIABLE SELECTION ON EUROSTA'S GALL SIZE. II. A PATH ANALYSIS OF THE ECOLOGICAL FACTORS BEHIND SELECTION

We examined phenotypic selection exerted by natural enemies on the gall-making fly Eurosta solidaginis in an extensive field study of 16 populations, spanning four generations. Gall-makers that induce small galls are vulnerable to the attack of Eurytoma gigantea. This imposes upward directional selection on gall size. Insectivorous birds, predominantly the downy woodpecker, are more likely to attack larvae that induce large galls than small ones, and this imposes downward directional selection. We used path analysis to explore the relative contributions of these natural enemies to the net directional selection on gall size. The path models further examined several ecological factors that influence selection intensity through their effects on parasitoid and bird attack rates. Net directional selection varied more strongly with E. gigantea attack than bird attack. Competitive interactions among birds and the three parasitoid species, including E. gigantea, were evidenced by low winter bird attack rates in fields where a high proportion of galls contained the overwintering parasitoids. Eurytoma gigantea attack was heavier in fields where mean gall size was small and bird attack heavier in fields where mean gall size was large. Neither birds nor E. gigantea showed simple density-dependent attack. Data suggested a form of frequency-dependent attack by birds but not by E. gigantea.     

Morphogenesis and gene expression in the soldier-caste differentiation of termites

Summary: Since almost all termite species possess a soldier caste, there must be ubiquitous mechanisms of soldier differentiation throughout isopteran species. In order to reveal the caste differentiation mechanisms, observations during the soldier morphogenesis and identification of soldier specific gene expression are thought to be important. In this article, I summarize research approaches for analyzing caste differentiation in termites, and introduce two of our studies in Hospitalitermes medioflavus (Termitidae) and Hodotermopsis japonica (Termopsidae).¶Colonies of the nasute termite H. medioflavus have soldiers with a frontal projection (nasus) on the head, from which defensive substances are secreted. During soldier differentiation from male minor worker to presoldier, the most dynamic morphogenesis occurs. In the presumptive nasus epithelium of minor workers, a disc-like structure termed "soldier-nasus disc" rapidly develops to form the nasus of presoldiers. This rapid growth is associated with two folding layers of cuticle and epithelium.¶To identify genes specifically expressed in soldiers of the damp wood termite Hodotermopsis japonica, a differential display using RT-PCR was tried, comparing mRNA from the heads of soldiers and pseudergates. An identified gene candidate termed SOL1 was expressed specifically in terminally differentiated mature soldiers, and the product of the gene was suggested to encode a novel protein with a putative signal peptide at the N-terminus. This gene was shown to be expressed in the mandibular glands which actually develop during the soldier differentiation. Thus, these molecular techniques are applicable to reveal the proximate mechanisms of caste determination in termites and other social insects.     

Plant polyploidy and host expansion in an insect herbivore

Polyploidization has played an essential role in the diversification of seed plants and often has profound effects on plant physiology and morphology. Yet, little is known about how plant polyploidization has shaped the ecology and evolution of interactions between phytophagous insects and their hosts. Polyploidization could either facilitate or impede colonization of new hosts. Greya politella (Lepidoptera: Prodoxidae) is highly specialized on plants in the genus Lithophragma (Saxifragaceae) throughout most of its geographic range. In central Idaho, some populations have shifted to the related Heuchera grossulariifolia, a plant that has repeatedly undergone autopolyploidization. Previous studies have shown that populations feeding natively on H. grossulariifolia prefer tetraploids to diploids in naturally mixed stands. We investigated whether this difference is caused by an inherent preference for tetraploids, or if the preference in present Heuchera-feeding populations has evolved over time. Moths from a strictly Lithophragma-feeding population were tested for preference of diploid or tetraploid H. grossulariifolia, using a combination of field experiments and caged choice trials. In all trials, attack rates on these non-hosts were very low, with no significant difference between ploidies. In addition, there was little evidence that females manipulated their clutch sizes when ovipositing into different plant species or ploidy levels. Hence, the local shift from Lithophragma to Heuchera in central Idaho is not due to failure of the moths to discriminate between these plant species. Furthermore, the higher attack rates on tetraploids in native H. grossulariifolia-feeding populations cannot be caused by a higher initial preference for these plants, but must instead be a result of differences in plant phenology and/or selection acting on local populations.     

Maternal care, defensive behavior, and sociality in neotropical Goniosoma harvestmen (Arachnida, Opiliones)

Summary: Goniosoma includes large and conspicuous species of harvestmen that occur exclusively in the Brazilian Atlantic forest. This paper reports on the parental activities, defensive behavior, and gregariousness of five species of Goniosoma, and also summarizes the published biological data for the genus. The behavioral patterns within the genus are discussed and a hypothesis for the emergence of gregariousness in the group is presented. Several Goniosoma live inside caves, mainly as trogloxenes. Although species of the genus comprise only 5% of the harvestmen fauna in Brazil, they account for 20.6% of all harvestmen species recorded in Brazilian caves. In addition to several morphological and physiological features that may favor the occupation of caves, species of Goniosoma also present subsocial behavior, which may confer a special advantage in this particular environment. Female protection is crucial for egg survival since predators may consume entire batches in a single night. Most species of Goniosoma form dense diurnal aggregations ranging from three to 200 individuals (mainly subadults and adults of both sexes). In a hypothetical scenario for the evolution of sociality in the Goniosoma, physiological constraints acting on individual harvestmen would lead to a behavioral response for the selection of sites with appropriate microclimatic conditions. The lack of cannibalism among adults and subadults may favor tolerance towards conspecifics, and may represent a pre-adaptation to gregariousness. The derived functions of gregarious behavior may include strengthening of the defensive signal through the collective release of a repugnant secretion, the dilution effect, and promptness in fleeing a predator attack as a consequence of the additional alarm role of scent gland secretions. These defensive functions may overcome the costs of group living and may be responsible for the maintenance of gregariousness in Goniosoma.