Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis

Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.     

The scaling of body size and mass in a host-parasitoid association: influence of host species and stage

We describe the allometry of body mass and body size as measured by hind-tibia length in males of Monoctonus paulensis (Ashmead) (Hymenoptera: Braconidae, Aphidiinae), a solitary parasitoid of aphids. To assess the influence of host quality on allometric relationships, we reared parasitoids on second and fourth nymphal instars of four different aphid species, Acyrthosiphon pisum (Harris), Macrosiphum creelii Davis, Myzus persicae (Sulzer) and Sitobion avenae (F.), under controlled conditions in the laboratory. Dry mass was positively correlated with hind-tibia length, and could be predicted from it, in unparasitized aphids, in aphid mummies containing parasitoid pupae, and in the parasitoid. The reduced-major-axis scaling exponents for the regression of dry mass on hind-tibia length were species-specific in aphids, reflecting differences in volume and shape between species. In mummified aphids, the stage at death influenced the size/mass relationship. In males of M. paulensis, the allometric exponent varied between parasitoids developing in different kinds of host. Individuals developing in pea aphid were absolutely larger in dry mass as well as proportionately larger relative to their hind-tibia length. We discuss the allometry of body size and body mass in relation to parasitoid fitness.     

Estimating population size and transmission bottlenecks in maternally transmitted endosymbiotic bacteria

Many species of bacterial endosymbionts are acquired by animal hosts before birth, through direct transmission from mothers to eggs or embryos. This vertical transmission imposes a reduction in numbers or "bottleneck," and the size of this bottleneck affects the population structure and evolution of symbiotic lineages. We have estimated the size of the transmission bottleneck in Buchnera, the bacterial symbiont of aphids, using basic light and electron microscopy techniques. By serial-sectioning whole aphid abdomens, their eggs, and embryos, we determined the following parameters: (i) The average size of a Buchnera cell is 2.9 mm in diameter. (ii) The total number of Buchnera in an Acyrthosiphon pisum embryo was around 36,700 whereas a first instar nymph contained more than 119,000. (iii) The number of symbionts per bacteriocyte was around 800 in an embryo and 3200 in a first instar nymph. (iv) The total number of Buchnera transmitted to each sexual egg ranged from 850 in Nasonovia to 1800 in A. pisum to more than 8000 in Uroleucon ambrosiae. (v) The total number of secondary endosymbionts in A. pisum was 12,170 for an embryo and 18,360 for a first instar nymph. Secondary symbionts were arranged both extracellularly and in clusters of 2000–8000 bacteria inside bacteriocytes. These numbers are consistent with the few previous estimates of symbiont population sizes based on counts of gene copies.     

Ultrastructure and transovarial transmission of endosymbiotic microorganisms in Palaeococcus fuscipennis (Burmeister) (Insecta, Hemiptera, Coccinea: Monophlebidae)

Ovaries ofPalaeococcus fuscipennis (Burmeister) are accompanied by large organs termed bacteriomes which are composed of large cells termed bacteriocytes. Each bacteriocyte is surrounded with small epithelial cells. The bacteriocyte cytoplasm is tightly packed with pleomorphic bacteria, whereas in epithelial cells small coccoid microorganisms are present. The number of coccoid bacteria is significantly lower than pleomorphic bacteria. The ovarioles containing choriogenic oocytes are invaded both by pleomorphic as well by coccoid bacteria. Microorganisms traverse the follicular epithelium and enter the perivitelline space. During advanced choriogenesis, endosymbionts are accumulated in the deep depression of the oocyte. Bacteria do not enter the ooplasm until the end of oocyte growth.     

Proton gradient mediates hemolymph trehalose influx into aphid bacteriocytes

Aphids harbor proteobacterial endosymbionts such as Buchnera aphidicola housed in specialized bacteriocytes derived from host cells. The endosymbiont Buchnera supplies essential amino acids such as arginine to the host cells and, in turn, obtains sugars needed for its survival from the hemolymph. The mechanism of sugar supply in aphid bacteriocytes has been rarely studied. It also remains unclear how Buchnera acquires its carbon source. The hemolymph sugars in Acyrthosiphon pisum are composed of the disaccharide trehalose containing two glucose molecules. Here, we report for the first time that trehalose is transported and used as a potential carbon source by Buchnera across the bacteriocyte plasma membrane via trehalose transporters. The current study characterized the bacteriocyte trehalose transporter Ap_ST11 (LOC100159441) using the Xenopus oocyte expression system. The Ap_ST11 transporter was found to be proton-dependent with a K_m value ≥700 mM. We re-examined the hemolymph trehalose at 217.8 mM using a fluorescent trehalose sensor. The bacteriocytes did not obtain trehalose by facilitated diffusion along the gradient across cellular membranes. These findings suggest that trehalose influx into the bacteriocytes depends on the extracellular proton-driven secondary electrochemical transporter.     

Buchnera aphidicola of the birch blister aphid,Hamamelistes betulinus (Horváth, 1896) (Insecta,Hemiptera, Aphididae: Hormaphidinae): molecular characterization,transmission between generations and its geographic significance

The birch blister aphid Hamamelistes betulinus, like most aphids, is host to obligate symbiotic bacterium Buchnera aphidicola. Ultrastructural and molecular analyses did not reveal the presence of secondary symbionts in the body of H. betulinus. The bacteria Buchnera aphidicola are transmitted to the next generation vertically (maternally). The bacteria released from the cytoplasm of the bacteriocyte to the haemolymph migrate to the embryo at the cellular blastoderm stage, through the opening at its posterior pole. Next, the bacteria enter the cytoplasm of newly formed bacteriocytes. The concept of the relationship between the geographic distribution of Hormaphidini aphids and the presence/absence of bacterium Buchnera aphidicola is discussed.     

Bacterial symbiosis maintenance in the asexually reproducing and regenerating flatworm Paracatenula galateia

Bacteriocytes set the stage for some of the most intimate interactions between animal and bacterial cells. In all bacteriocyte possessing systems studied so far, de novo formation of bacteriocytes occurs only once in the host development, at the time of symbiosis establishment. Here, we present the free-living symbiotic flatworm Paracatenula galateia and its intracellular, sulfur-oxidizing bacteria as a system with previously undescribed strategies of bacteriocyte formation and bacterial symbiont transmission. Using thymidine analogue S-phase labeling and immunohistochemistry, we show that all somatic cells in adult worms - including bacteriocytes - originate exclusively from aposymbiotic stem cells (neoblasts). The continued bacteriocyte formation from aposymbiotic stem cells in adult animals represents a previously undescribed strategy of symbiosis maintenance and makes P. galateia a unique system to study bacteriocyte differentiation and development. We also provide morphological and immunohistochemical evidence that P. galateia reproduces by asexual fragmentation and regeneration (paratomy) and, thereby, vertically transmits numerous symbiont-containing bacteriocytes to its asexual progeny. Our data support the earlier reported hypothesis that the symbiont population is subjected to reduced bottleneck effects. This would justify both the codiversification between Paracatenula hosts and their Candidatus Riegeria symbionts, and the slow evolutionary rates observed for several symbiont genes.     

Testing of Corner’s rules across woody plants in Tiantong region,Zhejiang Province: effects of individual density

Aims Corner’s rules reflect the architectural strategies of plants with respect to deployment of twig size and leaf size, as well as of the number of twigs and leaves. The objective of this study was to examine how Corner’s rules would vary among plants with different individual densities.
Methods The study site is located in the Tiantong National Forest Park (29.87° N, 121.65° E), Zhejiang Province. We measured twig cross-sectional area (twig size), total leaf area (leaf size per twig), and the number of twigs at a given twig size (branching intensity) in woody plants across 25 plots differing in stem density to examine the effects of individual competition on Corner’s rules. The standardized major axis (SMA) analysis was conducted to determine the quantitative relationships of twig size with leaf size and branching intensity.
Important findings Significant, positive allometric relationships between cross-sectional area and total leaf area were found in individual plants across all communities. There was no significant difference among communities of different density intervals in the slope of the linear regression between cross-sectional area and total leaf area of individual plants, and the common slope of the regressions was significantly greater than 1 (p < 0.001). The intercept was significantly greater for plants in communities with higher density than in those with lower density (p < 0.001), indicating that plants in a high density community support greater total leaf area than in a low density community for a given twig size. In contrast, a significant, negative allometric scaling relationship was found between branching intensity and cross-sectional area in individual plants across different communities. Also, nosignificant difference was found among plants in communities of different density intervals in the slope of the regression between branching intensity and cross-sectional area, and the common slope of the regressions was significantly less than –1 (p < 0.001). The intercept for the regression relationship between twig area and branching intensity was the same among plants in communities of different density intervals (p > 0.05), suggesting that plants in a high density community do not deploy more twigs per twig size than in a low density community. In summary, this study demonstrated that plants responded to changes in individual density by maintaining an invariant regression slope for the twig size-leaf size relationship and the twig size-branching intensity relationship, and that the Corner’s rules were not affected by individual density of the communities in the Tiantong region. However, changes in the intercept of the regression between twig size and leaf size indicate that deployment strategies between twig and leaf sizes could be adjusted with increasing individual plant competition, thus structuring species coexistence through niche differentiation.     

Allometric relations of total volumes of prolactin cells and corticotropic cells to body length in the annual cyprinodont Cynolebias whitei: effects of environmental salinity,stress and ageing

Summary An analysis of the allometric relations of the total volumes occupied by prolactin (PRL) and corticotropic (ACTH) cells (PRL volume and ACTH volume, respectively) to body length and a study of the immunocytochemical staining intensity of PRL and ACTH cells were used to determine the differences in activity of PRL and ACTH cells in freshwater-reared and in saltwater-reared Cynolebias whitei during the entire lifespan of this annual cyprinodont fish. An inflection in the allometric relation of PRL volume to body length was observed in fish of one-week old. The relatively large PRL volume in younger fish may be related to PRL cell activity before hatching. No inflections were observed in the allometric relations of PRL volume and ACTH volume to body length at the onset of maturation and the onset of ageing, indicating that the increased pituitary growth in maturing and ageing C. whitei is not the result of changes in PRL or ACTH cells. The slope of the allometric relation of PRL volume to body length in freshwater-reared fish was significantly steeper than the slope in saltwater-reared fish. The PRL volume in adult freshwater-reared fish was eight times larger than that in saltwater-reared fish of the same length. The intensity of immunocytochemical staining of saltwater PRL cells was significantly reduced. These volumetric and staining differences correspond to the low functional demand put upon PRL cells in saltwater-adapted fish. In contrast, the slope of the allometric relation of ACTH volume to body length and the intensity of immunocytochemical staining of ACTH cells were similar in freshwater-reared and in saltwater-reared fish. It is concluded that the functional demand put upon ACTH cells is similar in freshwater-reared and saltwater-reared C. whitei; the involvement of ACTH cells in the osmoregulation of the fish in both environments is similar.     

Genome expansion and differential expression of amino acid transporters at the aphid/Buchnera symbiotic interface

In insects, some of the most ecologically important symbioses are nutritional symbioses that provide hosts with novel traits and thereby facilitate exploitation of otherwise inaccessible niches. One such symbiosis is the ancient obligate intracellular symbiosis of aphids with the γ-proteobacteria, Buchnera aphidicola. Although the nutritional basis of the aphid/Buchnera symbiosis is well understood, the processes and structures that mediate the intimate interactions of symbiotic partners remain uncharacterized. Here, using a de novo approach, we characterize the complement of 40 amino acid polyamine organocation (APC) superfamily member amino acid transporters (AATs) encoded in the genome of the pea aphid, Acyrthosiphon pisum. We find that the A. pisum APC superfamily is characterized by extensive gene duplications such that A. pisum has more APC superfamily transporters than other fully sequenced insects, including a ten paralog aphid-specific expansion of the APC transporter slimfast. Detailed expression analysis of 17 transporters selected on the basis of their phylogenetic relationship to five AATs identified in an earlier bacteriocyte expressed sequence tag study distinguished a subset of eight transporters that have been recruited for amino acid transport in bacteriocyte cells at the symbiotic interface. These eight transporters include transporters that are highly expressed and/or highly enriched in bacteriocytes and intriguingly, the four AATs that show bacteriocyte-enriched expression are all members of gene family expansions, whereas three of the four that are highly expressed but not enriched in bacteriocytes retain one-to-one orthology with transporters in other genomes. Finally, analysis of evolutionary rates within the large A. pisum slimfast expansion demonstrated increased rates of molecular evolution coinciding with two major shifts in expression: 1) a loss of gut expression and possibly a gain of bacteriocyte expression and 2) loss of expression in all surveyed tissues in asexual females. Taken together, our characterization of nutrient AATs at the aphid/Buchnera symbiotic interface provides the first examination of the processes and structures operating at the interface of an obligate intracellular insect nutritional symbiosis, offering unique insight into the types of genomic change that likely facilitated evolutionary maintenance of the symbiosis.     

Testing of corner’s rules across woody plants in Tiantong region,Zhejiang Province: effects of micro-topography

Aims The size and quantity relationships between twigs and leaves can be used to describe the hydraulic properties of plants in response to environmental stresses. The objective of this study was to examine how twig-leaf relationship would vary with changes in micro-habitat conditions.
Methods The study site is located in the Tiantong National Forest Park (29.87° N, 121.65° E), Zhejiang Province. We measured twig cross-sectional area (twig size), sub-twig cross-sectional area (sub-twig size), individual leaf area, total leaf area (leaf size per twig), the number of twigs at a given twig size (twig intensity), and the number of leaves at a given twig size (leafing intensity) across individual woody plants on 10 plots in each of the convex and concave habitats within an evergreen broad-leaved forest. The standardized major axis (SMA) analysis was conducted to determine the scaling relationships between twig size and leaf size, between sub-twig size and twig intensity, and between leaf size and leafing intensity.
Important findings Significant, positive allometric relationships between twig cross-sectional area and total leaf area were found in plants in both types of micro-habitats (p < 0.001). There was no significant difference between the two micro-habitats in the slope of the regression between twig cross-sectional area and total leaf area, and the common slope of the regressions was significantly greater than 1 (p < 0.001). The intercept was significantlygreater in plants of the concave habitat than in plants of the convex habitat (p < 0.001), indicating that plants in a concave habitat support greater total leaf area at a given twig size than in a convex habitat. Significant, negative allometric scaling relationships were found between twig size and twig intensity in plants in both micro-habitats. There was also no significant difference between the two habitats in the slope of the regression between twig size and twig intensity, and the common slope of the regressions was significantly less than –1 (p < 0.001). The similar intercept in the regression relationship of twig area and twig intensity between the two habitats suggests that plants deploy similar amount of sub-twigs per twig size in both types of habitat. In addition, significant, negative allometric scaling relationships between leaf size and leafing intensity were found to be consistently conserved across micro-habitat types, with the common slope being smaller than –1. A higher value of y-intercept in the scaling relationships of leaf area vs. leafing intensity for plants in the concave habitat indicates that at a given leaf area, more leaves were supported by plants in a concave habitat than in a convex habitat. Overall, plants in a concave habitat tend to deploy more large leaves per twig size than those in a convex habitat. This study demonstrated that both the Corner’s rules and the leaf size-number trade-offs could be generalized to apply at the small local spatial scales. The magnitude and quantitative adjustment of twig-leaf deployment manifests a selection preference of hydraulic properties of plants in coping with changes in water availability between concave and convex habitats.     

Scaling of follicular sizes in mammalian ovaries

The scaling of ovarian follicle and oocyte sizes according to body weight ( M , ranging from 0005–500 kg) has been analysed using data obtained from 22 mammalian species in nine orders. The diameters of non-growing (primordial) follicles were correlated significantly with body weight, the relationship being described by the allometric formula y = 0028 M ^0.10. The mean size at which growing follicles began to accumulate extracellular fluid was approximately the same in all species, 0–3 mm diameter. Graafian follicle sizes varied allometrically with body weight as a result of differences in the volumes of follicular fluid rather than those of oocytes, which were relatively similar in eutherian mammals. The statistical significance of the correlation between Graafian and body sizes was increased when the dimensions for an ovulatory quota of follicles were combined because follicles in polyovulating species were disproportionately small. The total Graafian surface areas and volumes were then predicted from body weight by 58–4 M ^0.65 and 18–5 M ^1.06, respectively. Follicular dimensions in the three species of primates were significantly greater than predicted by the allometric relationship. The exponents of these relationships show that the total volume of a set of preovulatory follicles varies approximately isometrically with body weight and, therefore, with the presumptive hormone distribution volume ( M ^1.0). The hypoallometric relationship of follicular surface area demonstrates that, during the course of the evolution of body size, the surface area for secretion has not increased to match the dilution of hormones in the body pool.     

Evolution of brain–body allometry in Lake Tanganyika cichlids

Brain size is strongly associated with body size in all vertebrates. This relationship has been hypothesized to be an important constraint on adaptive brain size evolution. The essential assumption behind this idea is that static (i.e., within species) brain–body allometry has low ability to evolve. However, recent studies have reported mixed support for this view. Here, we examine brain–body static allometry in Lake Tanganyika cichlids using a phylogenetic comparative framework. We found considerable variation in the static allometric intercept, which explained the majority of variation in absolute and relative brain size. In contrast, the slope of the brain–body static allometry had relatively low variation, which explained less variation in absolute and relative brain size compared to the intercept and body size. Further examination of the tempo and mode of evolution of static allometric parameters confirmed these observations. Moreover, the estimated evolutionary parameters indicate that the limited observed variation in the static allometric slope could be a result of strong stabilizing selection. Overall, our findings suggest that the brain–body static allometric slope may represent an evolutionary constraint in Lake Tanganyika cichlids.     

Hominid femoral neck length

Human femoral neck length has a positive allometric or isometric relationship with total femoral length as shown by the reduced major axis slope based on logarithmic regression. The relatively long femoral neck lengths of Australopithecus cannot be explained solely as a consequence of small body size (contra Wolpoff, '78).     

Inbreeding depression, increased phenotypic variance, and a trade-off between gonads and appendages in selfed progeny of the aphid Prociphilus oriens

Trade-offs are potentially common among two or more traits whose development is dependent on the same resources. To detect genetic trade-offs, the techniques of quantitative genetics, pedigree analyses, and selection experiments have been used. This study demonstrates genetically based trade-offs between gonads and appendages in hatched larvae of the aphid Prociphilus oriens by focusing on enlarged variance among the families of selfed progeny. The selfed and outbred families were compared in respect to the size of morphological traits, gonad volume, and hatch dates as well as egg volume. Selfing not only increased the among-family variance component in all larval traits examined, but it also increased the mean size of all the morphological traits significantly. In contrast, gonad volume, a fitness component, was reduced with selfing. Calculation of the allometry (log-transformed regression) of larval traits to egg volume indicated that in the outbred group, morphological traits grew slowly relative to egg volume with slopes below 0.25, whereas gonads exhibited isometric growth. With selfing, most morphological traits had significantly steeper slopes, whereas the slope for gonads was greatly decreased. When the effect of egg volume was statistically removed from the means of selfed families, significant negative correlation was detected between the adjusted means of gonad volume and those of tibia length. This result suggests genetic trade-offs between gonad volume and tibia length. Thus, the evidence implies that at the loci governing the development of appendages, the dominant alleles function to canalize the development of tibiae into an optimal size, irrespective of egg volume. It is hypothesized that increased homozygosity of the deleterious recessive alleles reduced gonad volume through increasing the resource allocation to tibiae. The hypothesis of the gonad-appendage trade-off could be applied to explain the phenotypic evolution in some aphid species.     

Variation and covariation of life history traits in aphids are related to infection with the facultative bacterial endosymbiont Hamiltonella defensa

Host–symbiont associations play an important role in insects. In aphids, facultative symbionts affect host plant use and increase thermal tolerance and resistance to natural enemies. In spite of these beneficial effects on aphid fitness, the frequency of facultative symbionts in aphids ranges from low to intermediate. Tradeoffs induced by symbionts could prevent the fixation of symbionts in aphid populations. Therefore, we studied the life history traits and correlations between them in 21 clones of the black bean aphid, Aphis fabae, seven of which were infected with the facultative endosymbiont Hamiltonella defensa. We found that clones harbouring H. defensa exhibited significantly higher body mass at maturity and offspring production, and a marginally higher intrinsic rate of increase. However, development time and offspring body size did not differ between symbiont‐free and infected clones. In addition, body mass at maturity was positively correlated with offspring production, offspring body size and intrinsic rate of increase, whereas development time was negatively correlated with body mass at maturity, offspring production and offspring body size. Excluding infected clones had little effect on these correlations; only correlations between body mass at maturity and offspring production, and between development time and offspring body size, became nonsignificant. Therefore, we did not find any evidence for tradeoffs between life history traits induced by symbiont infection. In fact, infected clones had higher overall fitness than symbiont‐free clones under the conditions of our experiment, suggesting that symbionts do not impose costs on aphids harbouring them. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 237–247.     

Relative brain size,stratification, and social structure in anthropoids

The relationships between relative brain size and both stratification and social structure were examined in a total of 82 species of anthropoids. The species were divided into a total of 42 congeneric groups which consisted of congeneric species with similar ecologies and social structures. The relative brain size (RBS) was calculated for each congeneric group in each superfamily, based on an allometric equation describing the relationship between brain weight and body weight for each superfamily. Among congeneric groups with a common category of diet, RBS was significantly greater for terrestrial groups than for arboreal groups, and for polygynous (i.e. multi-female) groups than for monogynous (single-female) groups. Furthermore, RBS was significantly and positively correlated with the size of the home range per individual for the Cercopithecoidea, and with troop size for frugivorous groups of the Ceboidea. The results obtained suggest that factors associated with terrestriality and polygyny have been involved in the increases in relative brain size of anthropoids.