The development of sex differences in song in a tropical duetting wren.

The study of song development has focused on temperate zone birds in which typically only males sing. In the bay wren, Cantorchilus nigricapillus, both sexes sing, performing precisely timed, female-initiated duets in which birds alternate sex-specific song phrases. We investigated the development of these sex differences by collecting bay wren eggs and nestlings, and hand-raising them in individual acoustic isolation chambers. Each bird was tutored with either monophonic or stereophonic recordings of bay wren duets or heard no song. As adults, each tutored bird sang repertoires of complete duets, singing both male and female phrases. In addition, some birds sang only the male or female part of some duets to which they were exposed. Mono-tutored birds showed no sex-specificity in these solo songs, whereas stereo-tutored birds only sang solos consistent with their sex. In addition, stereo-tutored birds acquired songs over a longer period than did mono-tutored birds, and stereo-tutored females showed more sex-specificity than did males during early song production. Finally, we observed that tutored and acoustically isolated birds of both sexes invented male-like songs, whereas only males invent songs in the wild. These results reveal the relative roles of environmental versus innate influences in the development of sex-specific song in this species.

Community perspectives on the racial disparity in perinatal outcomes.

OBJECTIVE: The United States' infant and maternal mortality rates are significantly higher among non-Hispanic Black women and infants than women and infants of other races, independent of educational attainment or socioeconomic status. The purpose of this research was to understand conditions that lead to these disparities and propose practices for addressing them through community perspectives. METHOD: Researchers conducted six focus groups with African American women who had been pregnant previously (n = 27) and performed inductive thematic analysis looking at the interaction between race and health. RESULTS: Major themes included barriers to quality healthcare and support. Women perceived that healthcare professionals provided substandard care based on implicit biases and felt that asking questions of providers led to loss of autonomy.Conclusions and relevance: The perceived quality of a woman's perinatal experience is affected by women's relationships with their healthcare providers, their social support, and their sense of autonomy in decision-making. To improve the relationships between African American women and their providers, participants expressed that racism and implicit bias must be recognized and addressed. While this should be addressed in individual interactions, this study also suggests the role of policy change and system-level modifications that should be considered to effectively decrease the racial disparity in perinatal outcomes.

Blocking olfactory input alters aggression in male and female California mice (Peromyscus californicus).

Olfactory input into the brain can be disrupted by a variety of environmental factors, including exposure to pathogens or environmental contaminants. Olfactory cues are often eliminated in laboratory rats and mice through highly invasive procedures like olfactory bulbectomy, which may also disrupt accessory olfactory pathways and detection of non-volatile odors. In the present study, we tested whether inducing anosmia through intranasal infusion of zinc gluconate alters aggression in a monogamous, biparental rodent species, the California mouse (Peromyscus californicus). This less invasive method of manipulating olfaction selectively targets the olfactory epithelium and reduces the detection of volatile odors. Treatment with zinc gluconate extended the time required for male and female California mice to find hidden pieces of apple and reduced the amount of time spent investigating bedding that was soiled by unfamiliar males. Moreover, inhibition of olfaction with zinc gluconate reduced aggressiveness in both sexes as demonstrated by an increased attack latency in the resident-intruder test among same-sex dyads from the same treatment group. These results suggest that volatile olfactory cues are necessary for agonistic responses in both male and female California mice. Therefore, even in species with complex social systems that include territorial aggression and monogamy, volatile olfactory cues modulate agonistic behavior.

A successful model for longitudinal community-engaged health research: the 2040 Partners for Health Student Program.

Introduction: Community-based participatory research [CBPR] is an emerging approach to collaborative research aimed at creating locally effective and sustainable interventions. The 2040 Partners for Health student program was developed as a unique model of longitudinal CBPR. Analysis of this program and its components illuminates both the challenges and the opportunities inherent in community engagement. Methods: The program rests on a foundation of a community-based, non-profit organization and a supportive academic university centre. Inter-professional health students and community members of underserved populations work together on different health projects by employing an adapted CBPR methodology. Three successful examples of sustainable CBPR projects are briefly described. Results: The three projects are presented as primary outcomes resulting from this model. Benefits and challenges of the model as an approach to community-engaged research are discussed as well as secondary benefits of student participation. Conclusion: The 2040 Partners for Health student program represents a successful model of CBPR, illuminating common challenges and reiterating the profound value of community-engaged research.

Peromyscus as a model system for understanding the regulation of maternal behavior.

The genus Peromyscus has been used as a model system for understanding maternal behavior because of the diversity of reproductive strategies within this genus. This review will describe the ecological factors that determine litter size and litter quality in polygynous species such as Peromyscus leucopus and Peromyscus maniculatus. We will also outline the physiological and social factors regulating maternal care in Peromyscus californicus, a monogamous and biparental species. Because biparental care is relatively rare in mammals, most research in P. californicus has focused on understanding the biology of paternal care while less research has focused on understanding maternal care. As a result, the social, sensory, and hormonal cues used to coordinate parental care between male and female P. californicus have been relatively well-studied. However, less is known about the physiology of maternal care in P. californicus and in other Peromyscus species. The diversity of the genus Peromyscus provides the potential for future research to continue to examine how variation in social systems has shaped the mechanisms that underlie maternal care.

Vasopressin Proves Es-sense-tial: Vasopressin and the Modulation of Sensory Processing in Mammals.

As mammals develop, they encounter increasing social complexity in the surrounding world. In order to survive, mammals must show appropriate behaviors toward their mates, offspring, and same-sex conspecifics. Although the behavioral effects of the neuropeptide arginine vasopressin (AVP) have been studied in a variety of social contexts, the effects of this neuropeptide on multimodal sensory processing have received less attention. AVP is widely distributed through sensory regions of the brain and has been demonstrated to modulate olfactory, auditory, gustatory, and visual processing. Here, we review the evidence linking AVP to the processing of social stimuli in sensory regions of the brain and explore how sensory processing can shape behavioral responses to these stimuli. In addition, we address the interplay between hormonal and neural AVP in regulating sensory processing of social cues. Because AVP pathways show plasticity during development, early life experiences may shape life-long processing of sensory information. Furthermore, disorders of social behavior such as autism and schizophrenia that have been linked with AVP also have been linked with dysfunctions in sensory processing. Together, these studies suggest that AVP's diversity of effects on social behavior across a variety of mammalian species may result from the effects of this neuropeptide on sensory processing.

Efficient ΦC31 integrase-mediated site-specific germline transformation of Anopheles gambiae.

Current transgenic methodology developed for mosquitoes has not been applied widely to the major malaria vector Anopheles gambiae, which has proved more difficult to genetically manipulate than other mosquito species and dipteran insects. In this protocol, we describe ΦC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The ΦC31 system has many advantages over 'classical' transposon-mediated germline transformation systems, because it allows integration of large transgenes at specific, characterized genomic locations. Starting from a general protocol, we have optimized steps from embryo collection to co-injection of transgene-containing plasmid and in vitro-produced ΦC31 integrase mRNA. We also provide tips for screening transgenic larvae. The outlined procedure provides robust transformation in A. gambiae, resulting in homozygous transgenic lines in ∼2-3 months.

Transgenic Anopheles gambiae expressing an antimalarial peptide suffer no significant fitness cost.

Mosquito-borne diseases present some of the greatest health challenges faced by the world today. In many cases, existing control measures are compromised by insecticide resistance, pathogen tolerance to drugs and the lack of effective vaccines. In light of these difficulties, new genetic tools for disease control programmes, based on the deployment of genetically modified mosquitoes, are seen as having great promise. Transgenic strains may be used to control disease transmission either by suppressing vector populations or by replacing susceptible with refractory genotypes. In practice, the fitness of the transgenic strain relative to natural mosquitoes will be a critical determinant of success. We previously described a transgenic strain of Anopheles gambiae expressing the Vida3 peptide into the female midgut following a blood-meal, which exhibited significant protection against malaria parasites. Here, we investigated the fitness of this strain relative to non-transgenic controls through comparisons of various life history traits. Experiments were designed, as far as possible, to equalize genetic backgrounds and heterogeneity such that fitness comparisons focussed on the presence and expression of the transgene cassette. We also employed reciprocal crosses to identify any fitness disturbance associated with inheritance of the transgene from either the male or female parent. We found no evidence that the presence or expression of the effector transgene or associated fluorescence markers caused any significant fitness cost in relation to larval mortality, pupal sex ratio, fecundity, hatch rate or longevity of blood-fed females. In fact, fecundity was increased in transgenic strains. We did, however, observe some fitness disturbances associated with the route of inheritance of the transgene. Maternal inheritance delayed male pupation whilst paternal inheritance increased adult longevity for both males and unfed females. Overall, in comparison to controls, there was no evidence of significant fitness costs associated with the presence or expression of transgenes in this strain.

Contrasted Fitness Costs of Docking and Antibacterial Constructs in the EE and EVida3 Strains Validates Two-Phase Anopheles gambiae Genetic Transformation System.

The deployment of transgenic mosquitoes carrying genes for refractoriness to malaria has long been seen as a futuristic scenario riddled with technical difficulties. The integration of anti-malarial effector genes and a gene-drive system into the mosquito genome without affecting mosquito fitness is recognized as critical to the success of this malaria control strategy. Here we conducted detailed fitness studies of two Anopheles gambiae s.s. transgenic lines recently developed using a two-phase targeted genetic transformation system. In replicated cage-invasion experiments, males and females of the EE Phase-1 docking strain and EVida3 Phase-2 strain loaded with an antimicrobial peptide (AMP) expressed upon blood-feeding, were mixed with individuals of a recently-colonized strain of the Mopti chromosomal form. The experimental design enabled us to detect initial strain reproductive success differences, assortative mating and hybrid vigor that may characterize mosquito release situations. In addition, the potential fitness costs of the unloaded Phase-1 and loaded Phase-2 genetic constructs, independent of the strains' original genetic backgrounds, were estimated between the 1(st) instar larvae, pupae and adult stages over 10 generations. The Phase-1 unloaded docking cassette was found to have significantly lower allelic fitness relative to the wild type allele during larval development. However, overall genotypic fitness was comparable to the wild type allele across all stages leading to stable equilibrium in all replicates. In contrast, the Phase-2 construct expressing EVida3 disappeared from all replicates within 10 generations due to lower fitness of hemi- and homozygous larvae, suggesting costly background AMP expression and/or of the DsRed2 marker. This is the first study to effectively partition independent fitness stage-specific determinants in unloaded and loaded transgenic strains of a Phase-1-2 transformation system. Critically, the high fitness of the Phase-1 docking strain makes it the ideal model system for measuring the genetic load of novel candidate anti-malarial molecules in vivo.

Next-generation site-directed transgenesis in the malaria vector mosquito Anopheles gambiae: self-docking strains expressing germline-specific phiC31 integrase.

Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.

Naturally occurring variation in vasopressin immunoreactivity is associated with maternal behavior in female Peromyscus mice.

In many mammals, species-appropriate social behavior is necessary for an individual's ability to survive and reproduce. In the present study, we examined whether arginine-vasopressin (AVP) pathways that have been associated with social behavior differed between two closely related species of Peromyscus mice with different patterns of maternal behavior. We also tested whether individual levels of AVP-immunoreactive staining (AVP-ir) were associated with individual levels of maternal behavior as measured using a composite score consisting of huddling, nursing, grooming and time spent inside the nest (HNGI score). In addition, we examined whether these associations between vasopressin and behavior differed between species. Females from the highly biparental species, California mice, displayed higher AVP-ir in the bed nucleus of the stria terminalis (BNST), which corresponded with a higher level of nest building and a higher HNGI score than was found in the less parental white-footed mice. The HNGI score was positively associated with AVP-ir in the medial amygdala in female California mice but not white-footed mice. Finally, we examined whether AVP-ir in these pathways varied based on the species-specific rearing environments by reciprocally cross-fostering California mice and white-footed mice. In contrast to previous research with male California mice, cross-fostering itself had no effect on maternal behavior or any consistent effect on AVP-ir staining in brain areas such as the BNST and associated brain areas. This suggests that there is little plasticity in maternal behavior and that the underlying AVP system in females does not respond to the postnatal environment provided by the parents. The positive associations between maternal behavior and AVP-ir indicate that AVP may regulate maternal behavior despite the lack of plasticity in AVP and maternal behavior.

Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections.

Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the Streptomyces phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against Plasmodium yoelii nigeriensis, reducing average parasite intensity by 85%. Similar protection was observed against Plasmodium falciparum in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade.

Rational design of genetically stable, live-attenuated poliovirus vaccines of all three serotypes: relevance to poliomyelitis eradication.

The global eradication of poliomyelitis caused by wild-type virus is likely to be completed within the next few years, despite immense logistic and political difficulties, and may ultimately be followed by the cessation of vaccination. However, the existing live-attenuated vaccines have the potential to revert to virulence, causing occasional disease, and viruses can be shed by immunocompromised individuals for prolonged periods of time. Moreover, several outbreaks of poliomyelitis have been shown to be caused by viruses derived from the Sabin vaccine strains. The appearance of such strains depends on the prevailing circumstances but poses a severe obstacle to strategies for stopping vaccination. Vaccine strains that are incapable of reversion at a measurable rate would provide a possible solution. Here, we describe the constructions of strains of type 3 poliovirus that are stabilized by the introduction of four mutations in the 5' noncoding region compared to the present vaccine. The strains are genetically and phenotypically stable under conditions where the present vaccine loses the attenuating mutation in the 5' noncoding region completely. Type 1 and type 2 strains in which the entire 5' noncoding regions of Sabin 1 and Sabin 2 were replaced exactly with that of one of the type 3 strains were also constructed. The genetic stability of 5' noncoding regions of these viruses matched that of the type 3 strains, but significant phenotypic reversion occurred, illustrating the potential limitations of a rational approach to the genetic stabilization of live RNA virus vaccines.

Characterization of the promoters of Epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress.

Epsilon class GSTs (glutathione transferases) are expressed at higher levels in Anopheles gambiae mosquitoes that are resistant to DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] than in insecticide-susceptible individuals. At least one of the eight Epsilon GSTs in this species, GSTe2, efficiently metabolizes DDT to DDE [1,1-dichloro-2,2-bis-(p-chlorophenyl)ethane]. In the present study, we investigated the factors regulating expression of this class of GSTs. The activity of the promoter regions of GSTe2 and GSTe3 were compared between resistant and susceptible strains by transfecting recombinant reporter constructs into an A. gambiae cell line. The GSTe2 promoter from the resistant strain exhibited 2.8-fold higher activity than that of the susceptible strain. Six polymorphic sites were identified in the 352 bp sequence immediately upstream of GSTe2. Among these, a 2 bp adenosine indel (insertion/deletion) was found to have the greatest effect on determining promoter activity. The activity of the GSTe3 promoter was elevated to a lesser degree in the DDT-resistant strain (1.3-fold). The role of putative transcription-factor-binding sites in controlling promoter activity was investigated by sequentially deleting the promoter constructs. Several putative transcription-factor-binding sites that are responsive to oxidative stress were present within the core promoters of these GSTs, hence the effect of H2O2 exposure on the transcription of the Epsilon GSTs was investigated. In the DDT-resistant strain, expression of GSTe1, GSTe2 and GSTe3 was significantly increased by a 1-h exposure to H2O2, whereas, in the susceptible strain, only GSTe3 expression responded to this treatment.

Vasopressin and the transmission of paternal behavior across generations in mated, cross-fostered Peromyscus mice.

The role of arginine vasopressin (AVP) in the nongenomic transfer of paternal behavior from fathers to offspring was examined in Peromyscus. Male California mice (P. californicus) exposed to fewer retrievals by white-footed mouse (P. leucopus) foster parents displayed fewer retrievals of biological offspring. In contrast, white-footed mice were retrieved equally rarely by California mouse foster parents and by biological parents and displayed no changes in pup retrieval behavior. AVP-immunoreactive staining in the bed nucleus of the stria terminalis may predict paternal behavior because it correlated positively with retrievals and with a score consisting of huddling, grooming, and time inside the nest. The authors discuss AVP as a possible mechanism by which early experience shapes adult paternal behavior.

Simultaneous acceleration of the cell cycle and suppression of apoptosis by splice variant delta-6 of the candidate tumour suppressor LUCA-15/RBM5.

BACKGROUND: The short arm of chromosome 3 is thought to include one or more tumour suppressor genes (TSGs), since carcinoma of various tissues display deletions in this region. Many genes mapping to this region have recently been identified, including the LUCA-15/RBM5 gene. RESULTS: In this study we report the cloning from human bone marrow library of a splice variant of LUCA-15 which lacks exon 6, resulting in a frameshift and producing a truncated protein of 150 amino acids instead of 815 amino acids. This variant is widely expressed at a low level in normal tissues and is expressed at increased levels in T-leukaemic cell lines. Over-expression of this splice variant after electroporation both shortened the cell cycle and inhibited CD95-mediated apoptosis in CEM-C7 T-cells. In marked contrast, over-expression of the full length LUCA-15/RBM5 suppressed cell proliferation both by inducing apoptosis and by extending the G1 phase of the cell cycle. CONCLUSION: These results, taken together with previous observations from ourselves and others, suggest that LUCA-15 is involved in the control of both apoptosis and the cell cycle. Since oncogenesis often relies on separate changes in molecules regulating apoptosis on the one hand, and proliferation, on the other, the discovery of a candidate tumour suppressor gene which affects both processes simultaneously is likely to be of major significance.