Comparative evolutionary analyses of eight whitefly Bemisia tabaci sensu lato genomes: cryptic species, agricultural pests and plant-virus vectors.

BACKGROUND: The group of > 40 cryptic whitefly species called Bemisia tabaci sensu lato are amongst the world's worst agricultural pests and plant-virus vectors. Outbreaks of B. tabaci s.l. and the associated plant-virus diseases continue to contribute to global food insecurity and social instability, particularly in sub-Saharan Africa and Asia. Published B. tabaci s.l. genomes have limited use for studying African cassava B. tabaci SSA1 species, due to the high genetic divergences between them. Genomic annotations presented here were performed using the 'Ensembl gene annotation system', to ensure that comparative analyses and conclusions reflect biological differences, as opposed to arising from different methodologies underpinning transcript model identification. RESULTS: We present here six new B. tabaci s.l. genomes from Africa and Asia, and two re-annotated previously published genomes, to provide evolutionary insights into these globally distributed pests. Genome sizes ranged between 616-658 Mb and exhibited some of the highest coverage of transposable elements reported within Arthropoda. Many fewer total protein coding genes (PCG) were recovered compared to the previously published B. tabaci s.l. genomes and structural annotations generated via the uniform methodology strongly supported a repertoire of between 12.8-13.2 × 103 PCG. An integrative systematics approach incorporating phylogenomic analysis of nuclear and mitochondrial markers supported a monophyletic Aleyrodidae and the basal positioning of B. tabaci Uganda-1 to the sub-Saharan group of species. Reciprocal cross-mating data and the co-cladogenesis pattern of the primary obligate endosymbiont 'Candidatus Portiera aleyrodidarum' from 11 Bemisia genomes further supported the phylogenetic reconstruction to show that African cassava B. tabaci populations consist of just three biological species. We include comparative analyses of gene families related to detoxification, sugar metabolism, vector competency and evaluate the presence and function of horizontally transferred genes, essential for understanding the evolution and unique biology of constituent B. tabaci. s.l species. CONCLUSIONS: These genomic resources have provided new and critical insights into the genetics underlying B. tabaci s.l. biology. They also provide a rich foundation for post-genomic research, including the selection of candidate gene-targets for innovative whitefly and virus-control strategies.

A sucrose-specific receptor in Bemisia tabaci and its putative role in phloem feeding.

In insects, specialized feeding on the phloem sap (containing mainly the sugar sucrose) has evolved only in some hemipteran lineages. This feeding behavior requires an ability to locate feeding sites buried deeply within the plant tissue. To determine the molecular mechanism involved, we hypothesized that the phloem-feeding whitefly Bemisia tabaci relies on gustatory receptor (GR)-mediated sugar sensing. We first conducted choice assays, which indicated that B. tabaci adults consistently choose diets containing higher sucrose concentrations. Next, we identified four GR genes in the B. tabaci genome. One of them, BtabGR1, displayed significant sucrose specificity when expressed in Xenopus oocytes. Silencing of BtabGR1 significantly interfered with the ability of B. tabaci adults to discriminate between non-phloem and phloem concentrations of sucrose. These findings suggest that in phloem feeders, sugar sensing by sugar receptors might allow tracking an increasing gradient of sucrose concentrations in the leaf, leading eventually to the location of the feeding site.

Activation and detoxification of cassava cyanogenic glucosides by the whitefly Bemisia tabaci.

Two-component plant defenses such as cyanogenic glucosides are produced by many plant species, but phloem-feeding herbivores have long been thought not to activate these defenses due to their mode of feeding, which causes only minimal tissue damage. Here, however, we report that cyanogenic glycoside defenses from cassava (Manihot esculenta), a major staple crop in Africa, are activated during feeding by a pest insect, the whitefly Bemisia tabaci, and the resulting hydrogen cyanide is detoxified by conversion to beta-cyanoalanine. Additionally, B. tabaci was found to utilize two metabolic mechanisms to detoxify cyanogenic glucosides by conversion to non-activatable derivatives. First, the cyanogenic glycoside linamarin was glucosylated 1-4 times in succession in a reaction catalyzed by two B. tabaci glycoside hydrolase family 13 enzymes in vitro utilizing sucrose as a co-substrate. Second, both linamarin and the glucosylated linamarin derivatives were phosphorylated. Both phosphorylation and glucosidation of linamarin render this plant pro-toxin inert to the activating plant enzyme linamarase, and thus these metabolic transformations can be considered pre-emptive detoxification strategies to avoid cyanogenesis.

The molecular mechanisms that determine different degrees of polyphagy in the Bemisia tabaci species complex.

The whitefly Bemisia tabaci is a closely related group of >35 cryptic species that feed on the phloem sap of a broad range of host plants. Species in the complex differ in their host-range breadth, but the mechanisms involved remain poorly understood. We investigated, therefore, how six different B. tabaci species cope with the environmental unpredictability presented by a set of four common and novel host plants. Behavioral studies indicated large differences in performances on the four hosts and putative specialization of one of the species to cassava plants. Transcriptomic analyses revealed two main insights. First, a large set of genes involved in metabolism (>85%) showed differences in expression between the six species, and each species could be characterized by its own unique expression pattern of metabolic genes. However, within species, these genes were constitutively expressed, with a low level of environmental responsiveness (i.e., to host change). Second, within each species, sets of genes mainly associated with the super-pathways "environmental information processing" and "organismal systems" responded to the host switching events. These included genes encoding for proteins involved in sugar homeostasis, signal transduction, membrane transport, and immune, endocrine, sensory and digestive responses. Our findings suggested that the six B. tabaci species can be divided into four performance/transcriptomic "Types" and that polyphagy can be achieved in multiple ways. However, polyphagy level is determined by the specific identity of the metabolic genes/pathways that are enriched and overexpressed in each species (the species' individual metabolic "tool kit").

Glucosylation prevents plant defense activation in phloem-feeding insects.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

Molecular Evolution of the Glutathione S-Transferase Family in the Bemisia tabaci Species Complex.

The glutathione S-transferase (GST) family plays an important role in the adaptation of herbivorous insects to new host plants and other environmental constrains. The family codes for enzymes that neutralize reactive oxygen species and phytotoxins through the conjugation of reduced glutathione. Here, we studied the molecular evolution of the GST family in Bemisia tabaci, a complex of >35 sibling species, differing in their geographic and host ranges. We tested if some enzymes evolved different functionality, by comparing their sequences in six species, representing five of the six major genetic clades in the complex. Comparisons of the nonsynonymous to synonymous substitution ratios detected positive selection events in 11 codons of 5 cytosolic GSTs. Ten of them are located in the periphery of the GST dimer, suggesting a putative involvement in interactions with other proteins. Modeling the tertiary structure of orthologous enzymes, identified additional 19 mutations in 9 GSTs, likely affecting the enzymes' functionality. Most of the mutation events were found in the environmentally responsive classes Delta and Sigma, indicating a slightly different delta/sigma tool box in each species. At a broader genomic perspective, our analyses indicated a significant expansion of the Delta GST class in B. tabaci and a general association between the diet breadth of hemipteran species and their total number of GST genes. We raise the possibility that at least some of the identified changes improve the fitness of the B. tabaci species carrying them, leading to their better adaptation to specific environments.

A Transmissible RNA Pathway in Honey Bees.

Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive.

Species-complex diversification and host-plant associations in Bemisia tabaci: A plant-defence, detoxification perspective revealed by RNA-Seq analyses.

Insect-plant associations and their role in diversification are mostly studied in specialists. Here, we aimed to identify macroevolution patterns in the relationships between generalists and their host plants that have the potential to promote diversification. We focused on the Bemisia tabaci species complex containing more than 35 cryptic species. Mechanisms for explaining this impressive diversification have focused so far on allopatric forces that assume a common, broad, host range. We conducted a literature survey which indicated that species in the complex differ in their host range, with only few showing a truly broad one. We then selected six species, representing different phylogenetic groups and documented host ranges. We tested whether differences in the species expression profiles of detoxification genes are shaped more by their phylogenetic relationships or by their ability to successfully utilize multiple hosts, including novel ones. Performance assays divided the six species into two groups of three, one showing higher performance on various hosts than the other (the lower performance group). The same grouping pattern appeared when the species were clustered according to their expression profiles. Only species placed in the lower performance group showed a tendency to lower the expression of multiple genes. Taken together, these findings bring evidence for the existence of a common detoxification "machinery," shared between species that can perform well on multiple hosts. We raise the possibility that this "machinery" might have played a passive role in the diversification of the complex, by allowing successful migration to new/novel environments, leading, in some cases, to fragmentation and speciation.

Targeting detoxification genes by phloem-mediated RNAi: A new approach for controlling phloem-feeding insect pests.

Many phloem-feeding insects are considered severe pests of agriculture and are controlled mainly by chemical insecticides. Continued extensive use of these inputs is environmentally undesirable, and also leads to the development of insecticide resistance. Here, we used a plant-mediated RNA interference (RNAi) approach, to develop a new control strategy for phloem-feeding insects. The approach aims to silence "key" detoxification genes, involved in the insect's ability to neutralize defensive and toxic plant chemistry. We targeted a glutathione S-transferase (GST) gene, BtGSTs5, in the phloem-feeding whitefly Bemisia tabaci, a devastating global agricultural pest. We report three major findings. First, significant down regulation of the BtGSTs5 gene was obtained in the gut of B. tabaci when the insects were fed on Arabidopsis thaliana transgenic plants expressing dsRNA against BtGSTs5 under a phloem-specific promoter. This brings evidence that phloem-feeding insects can be efficiently targeted by plant-mediated RNAi. Second, in-silico and in-vitro analyses indicated that the BtGSTs5 enzyme can accept as substrates, hydrolyzed aliphatic- and indolic-glucosinolates, and produce their corresponding detoxified conjugates. Third, performance assays suggested that the BtGSTs5 gene silencing prolongs the developmental period of B. tabaci nymphs. Taken together, these findings suggest that BtGSTs5 is likely to play an important role in enabling B. tabaci to successfully feed on glucosinolate-producing plants. Targeting the gene by RNAi in Brassicaceae cropping systems, will likely not eliminate the pest populations from the fields but will significantly reduce their success over the growing season, support prominent activity of natural enemies, eventually allowing the establishment of stable and sustainable agroecosystem.

Glucosinolate Desulfation by the Phloem-Feeding Insect Bemisia tabaci.

Glucosinolates are plant secondary defense metabolites confined nearly exclusively to the order Brassicales. Upon tissue rupture, glucosinolates are hydrolyzed to various bioactive breakdown products by the endogenous plant enzyme myrosinase. As the feeding of chewing insect herbivores is associated with plant tissue damage, these insects have developed several independent strategies for coping with the glucosinolate-myrosinase defense system. On the other hand, our knowledge of how phloem-feeding insects interact with the glucosinolate-myrosinase system is much more limited. In fact, phloem feeders might avoid contact with myrosinase altogether so their susceptibility to intoxication by glucosinolate hydrolysis products is unclear. Previous studies utilizing Arabidopsis thaliana plants accumulating high levels of aliphatic- or indolic-glucosinolates indicated that both glucosinolate groups have moderate negative effects on the reproductive performance of Bemisia tabaci, a generalist phloem-feeding insect. To get a deeper understanding of the interaction between B. tabaci and glucosinolate-defended plants, adults were allowed to feed on artificial diet containing intact glucosinolates or on Brussels sprout and A. thaliana plants, and their honeydew was analyzed for the presence of possible metabolites. We found that B. tabaci is capable of cleaving off the sulfate group of intact glucosinolates, producing desulfoglucosinolates that cannot be activated by myrosinases, a mechanism described to date only in several chewing insect herbivores. The presence of desulfated glucosinolates in the honeydew of a generalist phloem-feeder may indicate the necessity to detoxify glucosinolates, likely due to some level of cellular damage during feeding, which results in glucosinolate activation, or as a mechanism to circumvent the non-enzymatic breakdown of indolic glucosinolates.

Exploring the role of juvenile hormone and vitellogenin in reproduction and social behavior in bumble bees.

BACKGROUND: The genetic and physiological pathways regulating behavior in solitary species are hypothesized to have been co-opted to regulate social behavior in social species. One classic example is the interaction between vitellogenin (an egg-yolk and storage protein) and juvenile hormone, which are positively correlated in most insect species but have modified interactions in highly eusocial insects. In some of these species (including some termites, ants, and the honey bee), juvenile hormone and vitellogenin levels are negatively correlated and juvenile hormone has shifted its role from a gonadotropin to a regulator of maturation and division of labor in the primarily sterile workers. The function of vitellogenin also seems to have broadened to encompass similar roles. Thus, the functions and molecular interactions of juvenile hormone and vitellogenin are hypothesized to have undergone changes during the evolution of eusociality, but the mechanisms underlying these changes are unknown.Bumble bees offer an excellent model system for testing how the relationship between juvenile hormone and vitellogenin evolved from solitary to social species. Bumble bee colonies are primitively eusocial and comprised of a single reproductive queen and facultatively sterile workers. In Bombus terrestris, juvenile hormone retains its ancestral role as a gonadotropin and is also hypothesized to regulate aggressive behavior. However, the function of vitellogenin and its interactions with juvenile hormone have not yet been characterized. RESULTS: By characterizing vitellogenin RNA expression levels (vg) in B. terrestris we show that vg is not associated with task and only partially associated with worker age, queen presence, and caste (queen vs worker). The correlations of vg with ovarian activation were not consistent across experiments, but both vg and ovarian activation were significantly associated with levels of aggression experienced by workers. Treatment with juvenile hormone did not affect vg levels in queenless groups. CONCLUSIONS: We suggest that social interactions affect vg levels more strongly than a worker's reproductive physiological state, and that juvenile hormone and vg are uncoupled in this species. Thus, although juvenile hormone maintains its traditional role as gonadotropin in B. terrestris, vg has already been co-opted into a novel role, consistent with the model that Bombus represents an intermediate stage in the evolution of eusociality.

Genomic analysis of the interactions between social environment and social communication systems in honey bees (Apis mellifera).

Social context is often a primary regulator of social behavior, but genes that affect or are affected by social context have rarely been investigated. In social insects, caste specific pheromones are key modulators of social behavior, e.g., in honey bees the queen mandibular gland (MG) pheromone mediates reproductive dominance, its absence prompting ovary activation and queen pheromone production in workers. Here, we investigate the effect of social environment on genome-wide expression patterns in the MG, to determine how social context modulates expression of genes that, in turn alter social environment. We used microarrays to examine the MGs of virgin and mated queens, and queenright (QR) and queenless (QL) workers with or without activated ovaries. Approximately 2554 transcripts were significantly differentially expressed among these groups, with caste and social context being the main regulators of gene expression patterns, while physiological state (ovary activation) only minimally affecting gene expression. Thus, social context strongly regulates expression of genes, which, in turn, shape social environment. Among these, 25 genes that are putatively involved in caste selective production of the fatty-acid derived MG pheromone were differentially expressed in queens and workers. These genes whose functions correspond with enzymatic or transport processes emphasize the occurrence of disparate pheromone biosynthetic pathways for queens and workers, adding another dimension regarding the regulation of these important pheromones. Gene ontology analysis also revealed genes of different functional categories whose expression was impacted by caste or by the social environment, suggesting that the MG has broader functions than pheromone biosynthesis.

Chemical profiles of two pheromone glands are differentially regulated by distinct mating factors in honey bee queens (Apis mellifera L.).

Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour's glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour's gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour's gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.

Activation of the Phenylpropanoid pathway in Nicotiana tabacum improves the performance of the whitefly Bemisia tabaci via reduced jasmonate signaling.

BACKGROUND: Phloem-feeding insects can manipulate plant-induced resistance and are able to suppress effective jasmonic acid/ethylene (JA/ET) defenses by the induction of inefficient salicylic acid (SA) based responses. As a result, activation of the phenylpropanoid biosynthesis pathway in transgenic plants is anticipated to cause complex interactions between phloem-feeding insects and their host plants due to predicted contradiction between two defense forces: the toxicity of various phenylpropanoids and the accumulation of SA via a branch of the activated pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated the effect of activating the phenylpropanoids pathway in Nicotiana tabacum, by over-expression of the PAP1 transcription factor, on the whitefly Bemisia tabaci, a phloem-feeding insect model. Our performance assays indicated that the over-expression made the transgenic plants a more suitable host for B. tabaci than wild-type (WT) plants, although these plants accumulated significantly higher levels of flavonoids. Transcription analyses of indicator genes in the SA (PR1a) and JA/ET (ERF1, COI1 and AOC) pathways followed by quantification of the SA and JA hormone levels, indicated that B. tabaci infestation periods longer than 8 hours, caused higher levels of activity of SA signaling in transgenic plants and higher levels of JA/ET signaling in WT plants. CONCLUSIONS/SIGNIFICANCE: Taken together, these results emphasize the important role JA/ET-induced defenses play in protecting plants from successful infestation by B. tabaci and likely other phloem-feeding insects. It also indicates the necessity of phloem feeders to suppress these defenses for efficient utilization of plant hosts. Our data also indicate that the defensive chemistry produced by the phenylpropanoids pathway has only a minor effect on the insect fitness.

Virgin honeybee queens fail to suppress worker fertility but not fertility signalling.

Queen mating status in social insects is a matter of crucial importance for workers because of its influence on the queen's productivity and consequently their fitness. Behavioural and physiological reactions of workers to the queens mating status have been studied as a proxy to mechanisms maintaining insect sociality. Here we show that unmated honeybee queens have considerably impaired capacity to trigger worker sterility and cooperative behaviour in comparison to mated (and thus more productive) queens and that under unmated queens social harmony in honeybee societies and queen's dominant position are somewhat compromised. Together with this it is shown that honeybee workers exposed to unmated queens despite being active reproductively and behaving accordingly display an impaired ability to advertise their fertility compared to queenless workers. These findings suggest that reproductive development, behavioural reactions and production of fertility signals are differentially regulated and differently influenced by the queen's presence.

Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology.

Honey bee colonies consist of tens of thousands of workers and a single reproductive queen that produces a pheromone blend which maintains colony organization. Previous studies indicated that the insemination quantity and volume alter queen mandibular pheromone profiles. In our 11-month long field study we show that workers are more attracted to high-volume versus low-volume inseminated queens, however, there were no significant differences between treatments in the number of queen cells built by workers in preparation for supersedure. Workers exposed to low-volume inseminated queens initiated production of queen-like esters in their Dufour's glands, but there were no significant difference in the amount of methyl farnesoate and juvenile hormone in worker hemolymph. Lastly, queen overwintering survival was unexpectedly lower in high-volume inseminated queens. Our results suggest that the queen insemination volume could ultimately affect colony health and productivity.

The role of tyramine and octopamine in the regulation of reproduction in queenless worker honeybees.

In honeybees, workers under queenless condition compete for reproduction and establish reproductive dominance hierarchy. Ovary activation is generally accompanied by the expression of queen-like pheromones. Biogenic amines (BAs), in particular dopamine, are believed to be involved in this process by regulating ovarian development. However, the role of BAs in establishing reproductive dominance or their effect on queen-like pheromone production was not investigated. Here, we explored the effect of octopamine (OA) and tyramine (TA) oral treatments on the propensity of treated bees to become reproductively dominant and produce queen-like pheromones in Dufour's and mandibular glands. One bee in a pair was treated with either OA or TA while the other was fed sugar solution. TA was found to enhance ovary development and the production of esters in the Dufour's gland and 9HDA (queen component) in the mandibular glands, thus facilitating worker reproductive dominance. OA, on the other hand, did not enhance ovarian development or ester production, but increased the production of 10HDA (worker major component) in the mandibular glands of their sugar-paired mates. OA is known to induce foraging behavior by workers, while increased production of 10HDA characterizes nursing workers. Therefore, we suggest that TA induces reproductive division of labor, while OA treatment results in caste differentiation of workers to foragers and nurses.

The gene road to royalty--differential expression of hydroxylating genes in the mandibular glands of the honeybee.

The advances in honeybee sociogenomics have paved the way for the study of social communication processes at the gene level, in particular the expression of caste-specific pheromones. The queen honeybee mandibular pheromone provides an excellent model system, in that biosynthesis of the hydroxylating fatty acid caste-specific pheromone appears to be reduced to a single chemical hydroxylation step of stearic acid. Queens are typified by omega-1-hydroxylation, as opposed to the worker-typical omega-hydroxylation. We hypothesized that this bifurcation is the consequence of differential expression of caste-specific genes that code for fatty acid-hydroxylating enzymes from the cytochrome P450 (CYP) family. Bioinformatics studies disclosed two candidate proteins CYP4AA1 and CYP18A1. We thus investigated the expression of these genes in the mandibular glands of queens, and of queenright (QR) and queenless (QL) workers. The real-time PCR results revealed that CYP4AA1 (omega-hydroxylation) was expressed at high levels in both QR and QL workers, whereas in queens its expression was negligible. The expression of CYP18A1 (omega-1-hydroxylation), on the other hand, was high in the queen's glands and negligible in those of QR workers. In QL workers, however, the expression of CYP18A1 was considerably elevated and significantly greater than in QR workers. Three-dimensional structural models constructed for these enzymes demonstrate differences in the active site between CYP18A1 and CYP4AA1, in line with their differential catalytic specificity. The fact that queen pheromone plasticity can be tracked all the way to gene expression provides a new insight into the process of caste differentiation and the accompanying social communication.

Uncoupling fertility from fertility-associated pheromones in worker honeybees (Apis mellifera).

Fertility-associated pheromones, chemical signals delineating ovarian development, were favourably selected in the course of evolution because it is in the best interest of both the signallers (in recruiting help from other colony members) and the receivers (in assisting them to reach an informed decision of how to maximize fitness). Such signals therefore should constitute honest, deception-proof indicators of ovarian development, suggesting, theoretically, that the processes of ovarian development and signal production are irreversibly coupled. Here we demonstrate that these processes can be uncoupled by treating queenless (QL) honeybee callow workers with methoprene, a juvenile hormone (JH) analog. While methoprene effectively inhibited ovarian development, it neither inhibited Dufour's fertility signal nor the mandibular glands' dominance signal. In fact, there was even a slight augmentation of both in the methoprene-treated bees. Thus, although fertility and fertility signals are tightly associated, they can be uncoupled by experimental manipulation. These results are consistent with the hypothesis that ovarian development and fertility-associated signal production are triggered by a common event/signal (e.g. queen pheromone disappearance) but comprise different regulatory systems. The evolutionary implication is that these two traits have evolved independently and may have been co-opted to emphasize the reproductive status of workers in the competition for reproduction.

The binding of fibrin sealant to collagen is influenced by the method of purification and the cross-linked fibrinogen-fibronectin (heteronectin) content of the 'fibrinogen' component.

Two fibrinogen preparations, each an intermediate in the manufacture of the 'fibrinogen' component of a commercial human tissue sealant, were made from a common cryoprecipitate source. The first preparation, prepared according to the process described by Schwartz et al. had a higher ratio of clottable to total protein than the second preparation, prepared according to that by Martinowitz and Bal but a much lower ratio of fibronectin to fibrinogen. After clotting with thrombin and solubilization and reduction of the clots, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a much higher content of high molecular weight polymers of fibrin(ogen) in the second preparation than in the first. The second preparation bound to collagen more strongly than did cryoprecipitate and much more strongly than did the first one. Experiments with highly purified proteins showed that fibronectin was essential in promoting progressive binding of fibrinogen to collagen under the action of activated factor XIII (transglutaminase). It was concluded that, because of their method of purification from cryoprecipitate, preparations of fibrinogen differ in their content of fibronectin and heteronectin. The binding of these proteins to collagen may improve the adhesion of tissue sealant clots to the extracellular matrix.