Managed and Wild Bee Flower Visitors and Their Potential Contribution to Pollination Services of Low-Chill Highbush Blueberry (Vaccinium corymbosum L.; Ericales: Ericaceae).

Highbush blueberry (Vaccinium corymbosum L.; Ericales: Ericaceae) is an important crop grown throughout the eastern United States and Canada. Cross-pollination by insects greatly enhances pollination and fruit set in highbush blueberry. In Florida, low-chill cultivars that flower during the winter when most bees are dormant are used, thus, making it difficult to utilize and depend on unmanaged bees. We investigated flower visitation rates by managed and wild bees and the subsequent berry formation, berry weight, and number of seeds/berry in highbush blueberry fields in north-central Florida. Additionally, we tested three pollinator treatments: 1) pollinator-excluded flowers, 2) open-pollinated treatments that were available to managed and wild bees, and 3) flowers that were hand pollinated. Overall, we found seven native bee species that contribute to highbush blueberry pollination in Florida, but managed honey bees and bumble bees were the main flower visitors. Additionally, 14.5 times more blueberries formed in the open treatments than in the pollinator exclusion treatments, thus illustrating the economic impact bees have on blueberry pollination. Most of the wild bees observed visiting blueberry flowers were ground-nesting species that need uncultivated areas for nesting sites. Therefore, leaving field edges uncultivated and some undisturbed habitat may increase native bee numbers within blueberry farms over time.

Managed Bumble Bees (Bombus impatiens) (Hymenoptera: Apidae) Caged With Blueberry Bushes at High Density Did Not Increase Fruit Set or Fruit Weight Compared to Open Pollination.

Highbush blueberry (Vaccinium corymbosum L.) is an important crop grown throughout Florida. Currently, most blueberry growers use honey bees (Apis mellifera L.) to provide pollination services for highbush blueberries even though bumble bees (Bombus spp.) have been shown to be more efficient at pollinating blueberries on a per bee basis. In general, contribution of bumble bees to the pollination of commercial highbush blueberries in Florida is unknown. Herein, we determined if managed bumble bees could contribute to highbush blueberry pollination. There were four treatments in this study: two treatments of caged commercial bumble bee (Bombus impatiens Cresson) colonies (low and high weight hives), a treatment excluding all pollinators, and a final treatment which allowed all pollinators (managed and wild pollinators) in the area have access to the plot. All treatments were located within a highbush blueberry field containing two cultivars of blooming plants, 'Emerald' and 'Millennia', with each cage containing 16 mature blueberry plants. We gathered data on fruit set, berry weight, and number of seeds produced per berry. When pollinators were excluded, fruit set was significantly lower in both cultivars (<8%) compared to that in all of the other treatments (>58%). Berry weight was not significantly different among the treatments, and the number of seeds per berry did not show a clear response. This study emphasizes the importance of bumble bees as an effective pollinator of blueberries and the potential beneficial implications of the addition of bumble bees in commercial blueberry greenhouses or high tunnels.

A two-color acid-free cartilage and bone stain for zebrafish larvae.

Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone, which negatively affects bone staining. We have developed an acid-free method to stain cartilage and bone simultaneously in zebrafish larvae. This method has the additional advantage that PCR genotyping of stained specimens is possible.

Neural crest patterning and the evolution of the jaw.

Here we present ideas connecting the behaviour of the cranial neural crest during development with the venerable, perhaps incorrect, view that gill-supporting cartilages of an ancient agnathan evolved into the skeleton of an early gnathostome's jaw. We discuss the pattern of migration of the cranial neural crest ectomesenchyme in zebrafish, along with the subsequent arrangement of postmigratory crest and head mesoderm in the nascent pharyngeal segments (branchiomeres), in diverse gnathostomes and in lampreys. These characteristics provide for a plausible von Baerian explanation for the problematic inside-outside change in topology of the gills and their supports between these 2 major groups of vertebrates. We consider it likely that the jaw supports did indeed arise from branchiomeric cartilages.

Specification and morphogenesis of the zebrafish larval head skeleton.

Forward genetic analyses can reveal important developmental regulatory genes and how they function to pattern morphology. This is because a mutated gene can produce a novel, sometimes beautiful, phenotype that, like the normal phenotype, immediately seems worth understanding. Generally the loss-of-function mutant phenotype is simplified from the wild-type one, and often the nature of the pattern simplification allows one to deduce how the wild-type gene contributes to patterning the normal, more complex, morphology. This truism seems no less valid for the vertebrate head skeleton than for other and simpler cases of patterning in multicellular plants and animals. To show this, we review selected zebrafish craniofacial mutants. "Midline group" mutations, in genes functioning in one of at least three signal transduction pathways, lead to neurocranial pattern truncations that are primarily along the mediolateral axis. Mutation of lazarus/pbx4, encoding a hox gene partner, and mutation of valentino/kreisler, a hox gene regulator, produce anterior-posterior axis disruptions of pharyngeal cartilages. Dorsoventral axis patterning of the same cartilages is disrupted in sucker/endothelin-1 mutants. We infer that different signal transduction pathways pattern cartilage development along these three separate axes. Patterning of at least the anterior-posterior and dorsoventral axes have been broadly conserved, e.g., reduced Endothelin-1 signaling similarly perturbs cartilage specification in chick, mouse, and zebrafish. We hypothesize that Endothelin-1 also is an upstream organizer of the patterns of cellular interactions during cartilage morphogenesis.

lazarus is a novel pbx gene that globally mediates hox gene function in zebrafish.

Individual vertebrate Hox genes specify aspects of segment identity along the anterior-posterior axis. The exquisite in vivo specificity of Hox proteins is thought to result from their interactions with members of the Pbx/Exd family of homeodomain proteins. Here, we report the identification and cloning of a zebrafish gene, lazarus, which is required globally for segmental patterning in the hindbrain and anterior trunk. We show that lazarus is a novel pbx gene and provide evidence that it is the primary pbx gene required for the functions of multiple hox genes during zebrafish development. lazarus plays a critical role in orchestrating the corresponding segmentation of the hindbrain and the pharyngeal arches, a key step in the development of the vertebrate body plan.

sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development.

Mutation of sucker (suc) disrupts development of the lower jaw and other ventral cartilages in pharyngeal segments of the zebrafish head. Our sequencing, cosegregation and rescue results indicate that suc encodes an Endothelin-1 (Et-1). Like mouse and chick Et-1, suc/et-1 is expressed in a central core of arch paraxial mesoderm and in arch epithelia, both surface ectoderm and pharyngeal endoderm, but not in skeletogenic neural crest. Long before chondrogenesis, suc/et-1 mutant embryos have severe defects in ventral arch neural crest expression of dHAND, dlx2, msxE, gsc, dlx3 and EphA3 in the anterior arches. Dorsal expression patterns are unaffected. Later in development, suc/et-1 mutant embryos display defects in mesodermal and endodermal tissues of the pharynx. Ventral premyogenic condensations fail to express myoD, which correlates with a ventral muscle defect. Further, expression of shh in endoderm of the first pharyngeal pouch fails to extend as far laterally as in wild types. We use mosaic analyses to show that suc/et-1 functions nonautonomously in neural crest cells, and is thus required in the environment of postmigratory neural crest cells to specify ventral arch fates. Our mosaic analyses further show that suc/et-1 nonautonomously functions in mesendoderm for ventral arch muscle formation. Collectively our results support a model for dorsoventral patterning of the gnathostome pharyngeal arches in which Et-1 in the environment of the postmigratory cranial neural crest specifies the lower jaw and other ventral arch fates.

islet reveals segmentation in the Amphioxus hindbrain homolog.

The vertebrate embryonic hindbrain is segmented into rhombomeres. Gene expression studies suggest that amphioxus, the closest invertebrate relative of vertebrates, has a hindbrain homolog. However, this region is not overtly segmented in amphioxus, raising the question of how hindbrain segmentation arose in chordate evolution. Vertebrate hindbrain segmentation includes the patterning of cranial motor neurons, which can be identified by their expression of the LIM-homeodomain transcription factor islet1. To learn if the amphioxus hindbrain homolog is cryptically segmented, we cloned an amphioxus gene closely related to islet1, which we named simply islet. We report that amphioxus islet expression includes a domain of segmentally arranged cells in the ventral hindbrain homolog. We hypothesize that these cells are developing motor neurons and reveal a form of hindbrain segmentation in amphioxus. Hence, vertebrate rhombomeres may derive from a cryptically segmented brain present in the amphioxus/vertebrate ancestor. Other islet expression domains provide evidence for amphioxus homologs of the pineal gland, adenohypophysis, and endocrine pancreas. Surprisingly, homologs of vertebrate islet1-expressing spinal motor neurons and Rohon-Beard sensory neurons appear to be absent.

The shaping of pharyngeal cartilages during early development of the zebrafish.

In zebrafish the cartilages of the pharynx develop during late embryogenesis and grow extensively in the larva before eventually being replaced by bone. Here we examine chondrocyte arrangements, shapes, numbers, and divisions in the young hyoid cartilages. We observe two distinct developmental phases, morphogenesis and growth. The first phase generates stereotypically oriented chondrocyte stacks that might form by intercalations among cells within the precartilage condensations. In mutants that have deformed cartilages the orientation of the stacks is changed, and we propose that their correct formation underlies the correct initial shaping of the organ. The following period of rapid, nearly isometric cartilage growth occurs by divisions of chondrocytes that are largely located near the joints, and appears to be under quite separate regulation.

An amphioxus snail gene: expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo.

Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, we have cloned and characterized the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates. We show that, at the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outgroup to all the characterized vertebrate snail genes. During embryogenesis snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. Our results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, our results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue.

Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm.

Zebrafish paraxial protocadherin (papc) encodes a transmembrane cell adhesion molecule (PAPC) expressed in trunk mesoderm undergoing morphogenesis. Microinjection studies with a dominant-negative secreted construct suggest that papc is required for proper dorsal convergence movements during gastrulation. Genetic studies show that papc is a close downstream target of spadetail, gene encoding a transcription factor required for mesodermal morphogenetic movements. Further, we show that the floating head homeobox gene is required in axial mesoderm to repress the expression of both spadetail and papc, promoting notochord and blocking differentiation of paraxial mesoderm. The PAPC structural cell-surface protein may provide a link between regulatory transcription factors and the actual cell biological behaviors that execute morphogenesis during gastrulation.

Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes.

Inhibition of fibroblast growth factor (FGF) signaling prevents trunk and tail formation in Xenopus and zebrafish embryos. While the T-box transcription factor Brachyury (called No Tail in zebrafish) is a key mediator of FGF signaling in the notochord and tail, the pathways activated by FGF in non-notochordal trunk mesoderm have been uncertain. Previous studies have shown that the spadetail gene is required for non-notochordal trunk mesoderm formation; spadetail mutant embryos have major trunk mesoderm deficiencies, but relatively normal tail and notochord development. We demonstrate here that spadetail encodes a T-box transcription factor with homologues in Xenopus and chick. Spadetail is likely to be a key mediator of FGF signaling in trunk non-notochordal mesoderm, since spadetail expression is regulated by FGF signaling. Trunk and tail development are therefore dependent upon the complementary actions of two T-box genes, spadetail and no tail. We show that the regulatory hierarchy among spadetail, no tail and a third T-box gene, tbx6, are substantially different during trunk and tail mesoderm formation, and propose a genetic model that accounts for the regional phenotypes of spadetail and no tail mutants.

Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm.

Cell fate decisions in early embryonic cells are controlled by interactions among developmental regulatory genes. Zebrafish floating head mutants lack a notochord; instead, muscle forms under the neural tube. As shown previously, axial mesoderm in floating head mutant gastrulae fails to maintain expression of notochord genes and instead expresses muscle genes. Zebrafish spadetail mutant gastrulae have a nearly opposite phenotype; notochord markers are expressed in a wider domain than in wild-type embryos and muscle marker expression is absent. We examined whether these two phenotypes revealed an antagonistic genetic interaction by constructing the double mutant. Muscle does not form in the spadetail;floating head double mutant midline, indicating that spadetail function is required for floating head mutant axial mesoderm to transfate to muscle. Instead, the midline of spadetail;floating head double mutants is greatly restored compared to that of floating head mutants; the floor plate is almost complete and an anterior notochord develops. In addition, we find that floating head mutant cells can make both anterior and posterior notochord when transplanted into a wild-type host, showing that enviromental signals can override the predisposition of floating head mutant midline cells to make muscle. Taken together, these results suggest that repression of spadetail function by floating head is critical to promote notochord fate and prevent midline muscle development, and that cells can be recruited to the notochord by environmental signals.

Genetic analysis of chromosomal rearrangements in the cyclops region of the zebrafish genome.

Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.

Equivalence in the genetic control of hindbrain segmentation in fish and mouse.

The vertebrate hindbrain is subdivided into a series of rhombomeres whose segmental organization serves to pattern the architecture and innervation of the developing head. The zebrafish gene valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and valentino mutants lack visible hindbrain segmentation caudal to the r3/4 boundary (Moens, C. B., Yan, Y.-L., Appel, B., Force, A. G., and Kimmel, C. B. (1996) Development 122, 3981-3990). Here we show that valentino is the zebrafish homologue of the mouse segmentation gene kreisler, which encodes a bZip transcription factor. The valentino gene is expressed in a manner consistent with its proposed role in subdividing rhombomeres 5 and 6 from their common precursor 'proto-segment' in the presumptive hindbrain, a process that we also demonstrate is reflected in the normal order of appearance of rhombomere boundaries. As well as having similar phenotypes with respect to visible hindbrain segmentation and patterns of marker gene expression, valentino and kreisler mutants have similar pharyngeal arch and inner ear defects, consistent with a conserved role for this gene in hindbrain segmentation and in patterning of the head periphery.

Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino.

The developing hindbrain is organized into a series of segments termed rhombomeres which represent lineage restricted compartments correlating with domains of gene expression and neuronal differentiation. In this study, we investigate the processes of hindbrain segmentation and the acquisition of segmental identity by analyzing the expression of zebrafish hox genes in the hindbrains of normal fish and fish with a loss-of-function mutation in the segmentation gene valentino (val, the homologue of mouse kreisler; Moens, C. B., Cordes, S. P. Giorgianni, M. W., Barsh, G. S. and Kimmel, C. B. (1998). Development 125, 381-391). We find that zebrafish hox genes generally have similar expression profiles to their murine and avian counterparts, although there are several differences in timing and spatial extent of expression which may underlie some of the functional changes that have occurred along the separate evolutionary lineages of teleosts and tetrapods. Our analysis of hox gene expression in val- embryos confirms that the val gene product is important for subdivision of the presumptive rhombomere 5 and 6 territory into definitive rhombomeres, suggests that the val gene product plays a critical role in regulating hox gene transcription, and indicates that some neural crest cells are inappropriately specified in val- embryos. Our analysis of gene expression at several developmental stages has allowed us to infer differences between primary and secondary defects in the val mutant: we find that extended domains of expression for some hox genes are secondary, late phenomena potentially resulting from inappropriate cell mixing or lack of normal inter-rhombomeric interactions in the caudal hindbrain.

Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo.

The head skeleton and muscles of the zebrafish develop in a stereotyped pattern in the embryo, including seven pharyngeal arches and a basicranium underlying the brain and sense organs. To investigate how individual cartilages and muscles are specified and organized within each head segment, we have examined their early differentiation using Alcian labeling of cartilage and expression of several molecular markers of muscle cells. Zebrafish larvae begin feeding by four days after fertilization, but cartilage and muscle precursors develop in the pharyngeal arches up to 2 days earlier. These chondroblasts and myoblasts lie close together within each segment and differentiate in synchrony, perhaps reflecting the interdependent nature of their patterning. Initially, cells within a segment condense and gradually become subdivided into individual dorsal and ventral structures of the differentiated arch. Cartilages or muscles in one segment show similar patterns of condensation and differentiation as their homologues in another, but vary in size and shape in the most anterior (mandibular and hyoid) and posterior (tooth-bearing) arches, possibly as a consequence of changes in the timing of their development. Our results reveal a segmental scaffold of early cartilage and muscle precursors and suggest that interactions between them coordinate their patterning in the embryo. These data provide a descriptive basis for genetic analyses of craniofacial patterning.

Genetic interactions in zebrafish midline development.

Mutational analyses have shown that the genes no tail (ntl, Brachyury homolog), floating head (flh, a Not homeobox gene), and cyclops (cyc) play direct and essential roles in the development of midline structures in the zebrafish. In both ntl and flh mutants a notochord does not develop, and in cyc mutants the floor plate is nearly entirely missing. We made double mutants to learn how these genes might interact. Midline development is disrupted to a greater extent in cyc;flh double mutants than in either cyc or flh single mutants; their effects appear additive. Both the notochord and floor plate are completely lacking, and other phenotypic disturbances suggest that midline signaling functions are severely reduced. On the other hand, trunk midline defects in flh;ntl double mutants are not additive, but are most often similar to those in ntl single mutants. This finding reveals that loss of ntl function can suppress phenotypic defects due to mutation at flh, and we interpret it to mean that the wild-type allele of ntl (ntl+) functions upstream to flh in a regulatory hierarchy. Loss of function of ntl also strongly suppresses the floor plate deficiency in cyc mutants, for we found trunk floor plate to be present in cyc;ntl double mutants. From these findings we propose that ntl+ plays an early role in cell fate choice at the dorsal midline, mediated by the Ntl protein acting to antagonize floor plate development as well as to promote notochord development.