Mass Transport Properties of the Rabbit Aortic Wall

Uptake of circulating macromolecules by the arterial wall may be a critical step in atherogenesis. Here we investigate the age-related changes in patterns of uptake that occur in the rabbit. In immature aortas, uptake was elevated in a triangle downstream of branch ostia, a region prone to disease in immature rabbits and children. By 16-22 months, uptake was high lateral to ostia, as is lesion prevalence in mature rabbits and young adults. In older rabbits there was a more upstream pattern, similar to the disease distribution in older people. These variations were predominantly caused by the branches themselves, rather than reflecting larger patterns within which the branches happened to be situated (as may occur with patterns of haemodynamic wall shear stress). The narrow streaks of high uptake reported in some previous studies were shown to be post mortem artefacts. Finally, heparin (which interferes with the NO pathway) had no effect on the difference in uptake between regions upstream and downstream of branches in immature rabbits but reversed the difference in older rabbits, as does inhibiting NO synthesis directly. Nevertheless, examination of uptake all around the branch showed that changes occurred at both ages and that they were quite subtle, potentially explaining why inhibiting NO has only minor effects on lesion patterns in mature rabbits and contradicting the earlier conclusion that mechanotransduction pathways change with age. We suggest that recently-established changes in the patterns of haemodynamic forces themselves are more likely to account for the age-dependence of uptake patterns.     

Elevated Uptake of Plasma Macromolecules by Regions of Arterial Wall Predisposed to Plaque Instability in a Mouse Model

Atherosclerosis may be triggered by an elevated net transport of lipid-carrying macromolecules from plasma into the arterial wall. We hypothesised that whether lesions are of the thin-cap fibroatheroma (TCFA) type or are less fatty and more fibrous depends on the degree of elevation of transport, with greater uptake leading to the former. We further hypothesised that the degree of elevation can depend on haemodynamic wall shear stress characteristics and nitric oxide synthesis. Placing a tapered cuff around the carotid artery of apolipoprotein E -/- mice modifies patterns of shear stress and eNOS expression, and triggers lesion development at the upstream and downstream cuff margins; upstream but not downstream lesions resemble the TCFA. We measured wall uptake of a macromolecular tracer in the carotid artery of C57bl/6 mice after cuff placement. Uptake was elevated in the regions that develop lesions in hyperlipidaemic mice and was significantly more elevated where plaques of the TCFA type develop. Computational simulations and effects of reversing the cuff orientation indicated a role for solid as well as fluid mechanical stresses. Inhibiting NO synthesis abolished the difference in uptake between the upstream and downstream sites. The data support the hypothesis that excessively elevated wall uptake of plasma macromolecules initiates the development of the TCFA, suggest that such uptake can result from solid and fluid mechanical stresses, and are consistent with a role for NO synthesis. Modification of wall transport properties might form the basis of novel methods for reducing plaque rupture.     

Unsteady and three-dimensional simulation of blood flow in the human aortic arch

A three-dimensional and pulsatile blood flow in a human aortic arch and its three major branches has been studied numerically for a peak Reynolds number of 2500 and a frequency (or Womersley) parameter of 10. The simulation geometry was derived from the three-dimensional reconstruction of a series of two-dimensional slices obtained in vivo using CAT scan imaging on a human aorta. The numerical simulations were obtained using a projection method, and a finite-volume formulation of the Navier-Stokes equations was used on a system of overset grids. Our results demonstrate that the primary flow velocity is skewed towards the inner aortic wall in the ascending aorta, but this skewness shifts to the outer wall in the descending thoracic aorta. Within the arch branches, the flow velocities were skewed to the distal walls with flow reversal along the proximal walls. Extensive secondary flow motion was observed in the aorta, and the structure of these secondary flows was influenced considerably by the presence of the branches. Within the aorta, wall shear stresses were highly dynamic, but were generally high along the outer wall in the vicinity of the branches and low along the inner wall, particularly in the descending thoracic aorta. Within the branches, the shear stresses were considerably higher along the distal walls than along the proximal walls. Wall pressure was low along the inner aortic wall and high around the branches and along the outer wall in the ascending thoracic aorta. Comparison of our numerical results with the localization of early atherosclerotic lesions broadly suggests preferential development of these lesions in regions of extrema (either maxima or minima) in wall shear stress and pressure.     

Dependence of adhesive behavior of neutrophils on local fluid dynamics in a region with recirculating flow

We have recently described patterns of adhesion of different types of leukocytes downstream of a backward facing step. Here the predicted fluid dynamics in channels incorporating backward facing steps are described, and related to the measured velocities of flowing cells, patterns of attachment and characteristics of rolling adhesion for neutrophils perfused over P-selectin. Deeper (upstream depth 300 microm, downstream depth 600 microm, maximum wall shear stress approximately 0.1 Pa) and shallower (upstream depth 260 microm, downstream depth 450 microm, maximum wall shear stress approximately 0.3 Pa) channels were compared. Computational fluid dynamics (CFD) predicted the presence of vortices downstream of the steps, distances to reattachment of flow, local wall shear stresses and components of velocity parallel and perpendicular to the wall. Measurements of velocities of perfused neutrophils agreed well with predictions, and suggested that adhesion to P-selectin should be possible in the regions of recirculating flow, but not downstream in re-established flow in the high shear channel. When channels were coated with a P-selectin-Fc chimaera, neutrophils were captured from flow and immobilised. Capture showed local maxima around the reattachment points, but was absent elsewhere in the high shear chamber. In the low shear chamber there was depression of adhesion just beyond the reattachment point because of expansion of flow and depletion of neutrophils near the wall. Inside the recirculation zones, adhesion decreased approaching the step because of an increasing, vertically upward velocity component. When channels were coated with P-selectin, neutrophils rolled in all regions, but lifted off the surface as they rolled backwards into low shear regions near the step. Rolling velocity in the recirculation zone was independent of shear stress, possibly because of the effects of vertical lift. We conclude that while local wall shear stress influences adhesive behavior, delivery of cells to the wall and their behavior after capture also depend on components of flow perpendicular to the wall.     

Growth patterns,and age and size at maturity in femaleCottus hangiongensis,with special reference to their life-history variation

Growth patterns of the 1982 year-class, individual growth patterns, age and size at sexual maturity and longevity in females of the river-sculpin,Cottus hangiongensis (Cottidae), were examined along the course of the Daitobetsu River of southern Hokkaido, Japan. Growth of females slightly varied both along the river course and among individual fishes: slow growth occurs in females from the lower reaches, while more rapid growth occurs in females from upstream areas. Body size and age at the first sexual maturity of females slightly increased towards the upstream, from 52 mm SL and 2 years in the most downstream area to 72 mm SL and 2–3 years in the uppermost site. Longevity was estimated to be 7 years in the downstream areas and 8–9 years in the upstream sites. These results suggest that female life history varies along the course of the river and thus allow us to consider the following alternative reproductive tactics: when females stay in the lower reaches, they attain sexual maturity at a smaller body size and younger age, and have a small clutch size, but when females migrate into the upper reaches, their maturity is delayed until they reach a larger body size and older age, and have a greater clutch size.     

Mechanisms initiating integrin-stimulated flow recruitment in arteriolar networks.

Our purpose was to investigate the local mechanisms involved in network-wide flow and diameter changes observed with localized downstream vitronectin receptor ligation; we tested specific K or Cl channels known to be involved in either dilation or elevated permeability following vitronectin receptor activation and tested integrin-linked pathway elements of tyrosine phosphorylation and protein kinase C (PKC). Arteriolar networks were observed in the cheek pouch tissue of anesthetized (pentobarbital sodium, 70 mg/kg) hamsters (n=86) using intravital microscopy. Terminal arteriolar branches of the networks were stimulated with micropipette LM609 (0.5-10 microg/ml, 60 s) alone or with inhibitors (separate micropipette). Hemodynamic changes (diameter, red blood cell flux, velocity) were observed at the upstream entrance to the network. LM609 alone stimulated first an increase in wall shear stress (WSS), followed by a dilation that recovered WSS to baseline or below. K channel inhibition (glybenclamide, 4-AP) had no effect on the initial peak in WSS, but decreased remote vasodilation. Cl channel inhibition (DIDS, IAA-94, niflumic acid) or inhibition of PKC (chelerythrine) prevented the initial peak in WSS and decreased remote vasodilation. Inhibition of tyrosine phosphorylation (genistein) prevented both. With the use of nitro-arginine at the observation site, the initial peak in WSS was not affected, but remote vasodilation was decreased. We conclude the remote response consists of an initial peak in WSS that relies on both PKC activity and depolarization downstream, leading to an upstream flow mediated dilation and a secondary remote dilation that relies on hyperpolarization downstream at the stimulus site; both components require tyrosine phosphorylation downstream.     

Real-time assessment of flow reversal in an eccentric arterial stenotic model

Plaque rupture is the leading cause of acute coronary syndromes and stroke. Plaque formation, otherwise known as stenosis, preferentially occurs in the regions of arterial bifurcation or curvatures. To date, real-time assessment of stenosis-induced flow reversal remains a clinical challenge. By interfacing microelectromechanical system (MEMS) thermal sensors with the high frequency pulsed wave (PW) Doppler ultrasound, we proposed to assess flow reversal in the presence of an eccentric stenosis. We developed a 3-D stenotic model (inner diameter of 6 mm, an eccentric stenosis with a height of 2.75 mm, and width of 21 mm) simulating a superficial arterial vessel. We demonstrated that heat transfer from the sensing element (2×80 μm²) to the flow field peaked as a function of flow rates at the throat of the stenosis along the center/midline of arterial model, and dropped downstream from the stenosis, where flow reversal was detected by the high frequency ultrasound device at 45 MHz. Computational fluid dynamics (CFD) codes are in agreement with the ultrasound-acquired flow profiles upstream, downstream, and at the throat of the stenosis. Hence, we characterized regions of eccentric stenosis in terms of changes in heat transfer along the midline of vessel and identified points of flow reversal with high spatial and temporal resolution.     

Variation in Constraint Versus Positive Selection as an Explanation for Evolutionary Rate Variation Among Anthocyanin Genes

It has been argued that downstream enzymes in metabolic pathways are expected to be subject to reduced selective constraint, while upstream enzymes, particularly those at pathway branch points, are expected to exhibit more frequent adaptive substitution than downstream enzymes. We examined whether these expectations are met for enzymes in the anthocyanin biosynthetic pathway in Ipomoea. Previous investigations have demonstrated that downstream enzymes in this pathway have substantially higher rates of nonsynonymous substitution than upstream enzymes. We demonstrate here that the difference in rates between the most upstream enzyme (CHS) and the two most downstream enzymes (ANS and UFGT) is explained almost entirely by differences in levels of selective constraint. Adaptive substitutions were not detected in any of these genes. Our results are consistent with suggestions that constraint is greater on enzymes with greater connectivity.     

The involvement of sex steroid hormones in downstream and upstream migratory behavior of masu salmon

From May through July when masu salmon, Oncorhynchus masou, commence downstream migration under natural conditions, yearling precocious male masu salmon (resident form) showed higher GSI and plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) in contrast to immature smolts (migratory form). From March through September coinciding with the upstream migration period, 2-year-old male and female adults also showed higher GSI and plasma levels of T, estradiol-17beta (E(2)) 11-KT, 17alpha-hydroxyprogesterone and 17alpha,20beta-dihydroxy-4-pregnene-3-one (DHP). In order to test the effects of steroid hormones on migratory behaviors, silascone tube capsules containing 500 microg of T, E(2), 11-KT, DHP, or a vehicle was implanted into smolts, castrated precocious males, or immature parr, and downstream and upstream behavior were observed in artificial raceways in spring and autumn. Downstream behavior of smolts was inhibited significantly by T, E(2) and 11-KT. Upstream behavior was stimulated by T and 11-KT in castrated precocious males and stimulated by T, E(2) and 11-KT in immature parr. These results indicate that T, E(2) and 11-KT are the factors regulating downstream and upstream migratory behavior. In particular, because of its changing patterns in plasma and significant effects, T, the common precursor hormone of E(2) (female) and 11-KT (male), is considered to play central roles in both types of behavior.     

Upstream-downstream movements of aquatic invertebrates in a Rocky Mountain stream

Simultaneous collections of drift and organisms moving either upstream or downstream in association with the substrate were made using a specially designed sampler. Samples were taken in a diel series along a transect across the study riffle of a Colorado foothills stream on six dates over an annual cycle. In addition to longitudinal movements, taxonomic composition and diel periodicity were evaluated. The insect-dominated fauna showed a net downstream displacement. Only the caddisflies Helicopsyche borealis and Hesperophylax occidentalis exhibited net upstream movement, primarily a result of low drift frequencies. The taxonomic composition of moving invertebrates differed from that of the benthos. Drift resembled downstream moving substrate-associated invertebrates in composition, but differed from that of the upstream directed fauna. Taxa collectively exhibited four types of diel patterns: 1) similar downstream (drift and substrate-associated movements) patterns, which generally differed from the upstream pattern; 2) similar benthic (upstream and downstream) patterns, which differed from that of drift; 3) aperiodic patterns; and 4) independent patterns for each type of directional movement. Analysis of size classes based on head capsule width for the mayfly Baetis tricaudatus showed significantly smaller size in stationary individuals compared with moving individuals in the population and revealed that nymphs moving during the day were smaller than those moving at night.     

Spatio-temporal changes of benthic macroinvertebrates in a cold Arkansas tailwater

This paper investigates spatial, seasonal and long-term changes in benthic macroinvertebrates in riffles of a cold tailwater. Cold tailwaters initially disrupt previously existing macroinvertebrate assemblages, but little is known about the long-term biological effects of a stable cold thermal regime. Assemblages at an upstream and downstream site of the Little Red River, Arkansas were investigated almost 30 years apart (1971 and 1999). Based upon published literature demonstrating the stability of benthic assemblages within unaltered environments, we predicted that the assemblages would be similar for each variable investigated. The benthic macroinvertebrate assemblages can be characterized as low diversity, with a total of 17 taxa identified. Isopods and Diptera comprised ~80% of all individuals. Other than chironomids, insects and particularly EPT taxa were poorly represented. Recent macroinvertebrate densities were significantly greater compared to the historical study period for the downstream site. Assemblage comparisons revealed moderate differences between study periods. Macroinvertebrate density was significantly greater upstream than downstream in the 1971 study period, yet taxa richness was significantly greater downstream for both study periods. Faunal composition was significantly different for upstream and downstream sites. Seasonal differences in numerical standing crop were identified for the 1971 upstream and 1999 downstream data sets. Low to moderate levels of seasonal, spatial and historical variation among benthic macroinvertebrate assemblages were attributed to environmental (temperature and flow) stability. The lack of aquatic insects other than chironomids over a 30-year period is indicative of the extreme constraints placed upon insect development within this cold regulated river.     

Hemodynamics simulation and identification of susceptible sites of atherosclerotic lesion formation in a model abdominal aorta

Employing the rabbit's abdominal aorta as a suitable atherosclerotic model, transient three-dimensional blood flow simulations and monocyte deposition patterns were used to evaluate the following hypotheses: (i) simulation of monocyte transport through a model of the rabbit abdominal aorta yields cell deposition patterns similar to those seen in vivo, and (ii) those deposition patterns are correlated with hemodynamic wall parameters related to atherosclerosis. The deposition pattern traces a helical shape down the aorta with local elevation in monocyte adhesion around vessel branches. The cell deposition pattern was altered by an exercise waveform with fewer cells attaching in the upper abdominal aorta but more attaching around the renal orifices. Monocyte deposition was correlated with the wall shear stress gradient and the wall shear stress angle gradient. The wall stress gradient, the wall shear stress angle gradient and the normalized monocyte deposition fraction were correlated with the distribution of monocytes along the abdominal aorta and monocyte deposition is correlated with the measured distribution of monocytes around the major abdominal branches in the cholesterol-fed rabbit. These results suggest that the transport and deposition pattern of monocytes to arterial endothelium plays a significant role in the localization of lesions.     

Flow fields generated by peristaltic reflex in isolated guinea pig ileum: impact of contraction depth and shoulders

The guinea pig ileum responds to distension with characteristic wall movements, luminal pressure gradients, and outflow (the peristaltic reflex). To date, little is known about whether the peristaltic reflex generates flow events other than laminar flow. Here we used a numerical method to solve for the flow generated by moving walls to assess occlusive contractions (case 1), nonocclusive contractions (case 2), and contractions with steep shoulders (case 3) for which visual parameters of wall movements are published. We found that all three contraction cases produced pressure differentials across the coapting segment, downstream and reverse flow, and vortical flow patterns that redistributed particles and mixed liquids. Contractions generated pressures and shear stresses, particularly along the moving section of the wall. The nonocclusive contraction was much less effective than the occlusive contraction with the steep shoulders; the occlusive contraction with flat shoulders had an intermediate effect. Our analysis shows that even peristaltic contractions produce not only laminar flow but also many flow events likely to promote digestion and absorption. The visual patterns of contractions impact the patterns of luminal flow, and precise definition of wall movements is critical to quantify the fluid mechanical consequences of intestinal contractions.     

Regulation of midline development by antagonism of lefty and nodal signaling.

The embryonic midline is crucial for the development of embryonic pattern including bilateral symmetry and left-right asymmetry. In zebrafish, lefty1 (lft1) and lefty2 (lft2) have distinct midline expression domains along the anteroposterior axis that overlap with the expression patterns of the nodal-related genes cyclops and squint. Altered expression patterns of lft1 and lft2 in zebrafish mutants that affect midline development suggests different upstream pathways regulate each expression domain. Ectopic expression analysis demonstrates that a balance of lefty and cyclops signaling is required for normal mesendoderm patterning and goosecoid, no tail and pitx2 expression. In late somite-stage embryos, lft1 and lft2 are expressed asymmetrically in the left diencephalon and left lateral plate respectively, suggesting an additional role in laterality development. A model is proposed by which the vertebrate midline, and thus bilateral symmetry, is established and maintained by antagonistic interactions among co-expressed members of the lefty and nodal subfamilies of TGF-beta signaling molecules.     

Temperature-sensitive anthocyanin production in flowers of Plantago lanceolata

Flower color in the weedy perennial Plantago lanceolata is phenotypically plastic. Darker flowers are produced at cooler ambient temperatures, and circumstantial evidence suggests that this is adaptive. The goal of this project was to investigate the chemical basis for the color plasticity. To test the hypothesis that increased anthocyanin production at low temperatures underlies the plasticity, extracts of P. lanceolata flowers produced at warm and cool temperatures were analyzed using UV/visible spectrophotometry coupled with mass spectrometry. Mass spectrometry allowed us to compare relative abundances of individual anthocyanins. Seventeen anthocyanins, derived from both cyanidin and delphinidin branches of the anthocyanin biosynthetic pathway, were detected. Most of these significantly increased in abundance under cool conditions. Genotypes differed significantly in anthocyanin levels and in their sensitivity to temperature change. Genotypes that showed greater floral color plasticity tended to show also greater temperature sensitivity with respect to anthocyanin production. Data suggest that the temperature regulation of the anthocyanin biosynthetic pathway occurs both upstream and downstream of the divergence of the cyanidin and delphinidin branches. The degree of temperature sensitivity, i.e. phenotypic plasticity, appears to be controlled downstream, whereas the overall temperature effect appears to be controlled upstream.     

Computational analysis of coupled blood-wall arterial LDL transport

The transport of macromolecules, such as low density lipoproteins (LDLs), across the artery wall and their accumulation in the wall is a key step in atherogenesis. Our objective was to model fluid flow within both the lumen and wall of a constricted, axisymmetric tube simulating a stenosed artery, and to then use this flow pattern to study LDL mass transport from the blood to the artery wall. Coupled analysis of lumenal blood flow and transmural fluid flow was achieved through the solution of Brinkman's model, which is an extension of the Navier-Stokes equations for porous media. This coupled approach offers advantages over traditional analyses of this problem, which have used possibly unrealistic boundary conditions at the blood-wall interface; instead, we prescribe a more natural pressure boundary condition at the adventitial vasa vasorum, and allow variations in wall permeability due to the occurrence of plaque. Numerical complications due to the convection dominated mass transport process (low LDL diffusivity) are handled by the streamline upwind/Petrov-Galerkin (SUPG) finite element method. This new fluid-plus-porous-wall method was implemented for conditions typical of LDL transport in a stenosed artery with a 75 percent area reduction (Peclet number=2 x 10(8)). The results show an elevated LDL concentration at the downstream side of the stenosis. For the higher Darcian wall permeability thought to occur in regions containing atheromatous lesions, this leads to an increased transendothelial LDL flux downstream of the stenosis. Increased transmural filtration in such regions, when coupled with a concentration-dependent endothelial permeability to LDL, could be an important contributor to LDL infiltration into the arterial wall. Experimental work is needed to confirm these results.     

Comparative analysis of orthologous eukaryotic mRNAs: potential hidden functional signals

Sequencing of multiple, nearly complete eukaryotic genomes creates opportunities for detecting previously unnoticed, subtle functional signals in non-coding regions. A genome-wide comparative analysis of orthologous sets of mammalian and yeast mRNAs revealed distinct patterns of evolutionary conservation at the boundaries of the untranslated regions (UTRs) and the coding region (CDS). Elevated sequence conservation was detected in ~30 nt regions around the start codon. There seems to be a complementary relationship between sequence conservation in the ~30 nt regions of the 5′-UTR immediately upstream of the start codon and that in the synonymous positions of the 5′-terminal 30 nt of the CDS: in mammalian mRNAs, the 5′-UTR shows a greater conservation than the CDS, whereas the opposite trend holds for yeast mRNAs. Unexpectedly, a ~30 nt region downstream of the stop codon shows a substantially lower level of sequence conservation than the downstream portions of the 3′-UTRs. However, the sequence in this poorly conserved 30 nt portion of the 3′-UTR is non-random in that it has a higher GC content than the rest of the UTR. It is hypothesized that the elevated sequence conservation in the region immediately upstream of the start codon is related to the requirement for initiation factor binding during pre-initiation ribosomal scanning. In contrast, the poorly conserved region downstream of the stop codon could be involved in the post- termination scanning and dissociation of the ribosomes from the mRNA, which requires only the mRNA–ribosome interaction. Additionally, it was found that the choice of the stop codon in mammals, but not in yeasts, and the context in the immediate vicinity of the stop codons in both mammals and yeasts are subject to strong selection. Thus, genome-wide analysis of orthologous gene sets allows detection of previously unrecognized patterns of sequence conservation, which are likely to reflect hidden functional signals, such as ribosomal filters that could regulate translation by modulating the interaction between the mRNA and ribosomes.     

Conducted dilations initiated by purines in arterioles are endothelium dependent and require endothelial Ca2+

The signaling pathways underlying the regulation of vascular resistance by purines in intact microvessels and particularly in communication of remote vasomotor responses are unclear. One process by which remote regions of arterioles communicate is via transmission of signals axially along the vessel wall. In this study, we identified a pathway for local and conducted dilations initiated by purines. Adenosine (Ado) or ATP (bind P1 and P2 purinergic receptors, respectively) was micropipette applied to arterioles (maximum diameter approximately 40 microm) in the cheek pouch of anesthetized hamsters. Observations were made at the site of stimulation (local) or approximately 1200 microm upstream along the same vessel. P2 antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium and suramin) inhibited local constriction to ATP, whereas local and upstream dilations were unaffected. In contrast, during inhibition of P1 receptors (with xanthine amine congener) the local constriction was unchanged, whereas both local and upstream dilations to ATP were inhibited. Hydrolysis of ATP to Ado is implicated in the dilator response as blocking 5'-ectonucleotidase (with alpha,beta-methyleneadenosine 5'-diphosphate) attenuated ATP-induced dilations. After endothelium denudation, constriction to ATP was unchanged, but dilations to both ATP and Ado were inhibited, identifying endothelial cells (ECs) as the primary target for P1-mediated dilation. Purines increased EC Ca2+ locally and upstream. Chelation of EC Ca2+ (with BAPTA) abolished the local and upstream dilations to P1 receptor stimulation. Collectively, these data demonstrate that stimulation of P1 receptors on ECs produces a vasodilation that spreads to remote regions. There is an associated increase in EC Ca2+, which is a required signaling intermediate in the manifestation of both the local and axially communicated arteriolar dilations.     

Genome-wide polymorphisms show unexpected targets of natural selection

Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species' wide latitudinal range. We examined 9112 polymorphic loci from upstream non-coding and coding regions of genes for signatures of selection with respect to gene function and tissue- and ontogenetic gene expression. We found that genetic differentiation (F(ST)) varied significantly across functional gene classes. The strongest enrichment occurred in the upstream regions of E3 ligase genes, enzymes known to regulate protein abundance during development and environmental stress. We found enrichment for high heterozygosity in genes directly involved in immune response, particularly NALP genes, which mediate pro-inflammatory signals during bacterial infection. We also found higher heterozygosity in immune genes in the southern population, where disease incidence and pathogen diversity are greater. Similar to the major histocompatibility complex in mammals, balancing selection may enhance genetic diversity in the innate immune system genes of this invertebrate. Overall, our results show that how genome-wide polymorphism data coupled with growing databases on gene function and expression can combine to detect otherwise hidden signals of selection in natural populations.