Non-primate Species Research Articles

Vocal functional flexibility in a nonprimate vocal learning species

Abstract Can nonhuman animals use the same acoustic signal to transmit different illocutions on different occasions? This communicative capacity is known as vocal functional flexibility and occurs, for example, in speech, when a sentence serves different illocutionary forces or functions on different occasions based on changes to visual and intonational cues. Although common in human speech, there is a lack of clear evidence for this ability in other species. Here, we examined a likely candidate, the Brown-headed Cowbird (Molothrus ater), which is a vocal-learning songbird species that develops a repertoire of structurally distinct song types. Most of this species’ songs are directed towards conspecific males and females less than a meter away, making it unusually easy to determine the apparent target of songs, unlike the broadcast songs done by most songbirds. Songs directed to other males have clear aggressive/threatening intent, while those to females involve courtship/sexual intent. Extensive prior work shows that male cowbirds perform the visual display that accompanies singing differently in these two social settings and also modulate the intonation of song types differently. Because of these display and tonal modulations, constancy of song type usage across male- vs female-directed singing would provide evidence of vocal functional flexibility. Herein, we examined 4,828 songs in three captive flocks containing twenty-four males and thirty females during the breeding season. Males did not use their song types randomly and had strongly favored songs and less commonly used ones. Importantly, favored song types and less commonly used ones were the same whether directing courtship song to a female, aggressive song to another male or singing nonsocially with no receiver nearby. Results were consistent within and across the three flocks, providing strong evidence of vocal functional flexibility. These findings indicate that some species may evolve the ability to modulate and exaggerate visual display components and prosody more than vocal presentation per se because a learned phonological system in this and possibly other species is constrained by its vital role as an indicator trait.

Serial visual reversal learning in captive black-handed spider monkeys, Ateles geoffroyi

Recent research suggests that socio-ecological factors such as dietary specialization and social complexity may be drivers of advanced cognitive skills among primates. Therefore, we assessed the ability of 12 black-handed spider monkeys (Ateles geoffroyi), a highly frugivorous platyrrhine primate with strong fission-fusion dynamics, to succeed in a serial visual reversal learning task. Using a two-alternative choice paradigm we first trained the animals to reliably choose a rewarded visual stimulus over a non-rewarded one. Upon reaching a pre-set learning criterion we then switched the reward values of the two stimuli and assessed if and how quickly the animals learned to reverse their choices, again to a pre-set learning criterion. This stimulus reversal procedure was then continued for a total of 80 sessions of 10 trials each. We found that the spider monkeys quickly learned to reliably discriminate between two simultaneously presented visual stimuli, that they succeeded in a visual reversal learning task, and that they displayed an increase in learning speed across consecutive reversals, suggesting that they are capable of serial reversal learning-set formation with visual cues. The fastest-learning individual completed five reversals within the 80 sessions. The spider monkeys outperformed most other primate and nonprimate mammal species tested so far on this type of cognitive task, including chimpanzees, with regard to their learning speed in both the initial learning task and in the first reversal task, suggesting a high degree of behavioral flexibility and inhibitory control. Our findings support the notion that socio-ecological factors such as dietary specialization and social complexity foster advanced cognitive skills in primates.

Human rod photoreceptor outer segments are supported by accessory inner segment structures.

The first steps in vision take place in photoreceptor cells, which are highly compartmentalized neurons exhibiting significant structural variation across species. The light-sensitive ciliary compartment, called the outer segment, is located atop of the cell soma, called the inner segment. In this study, we present an ultrastructural analysis of human photoreceptors, which reveals that, in contrast to this classic arrangement, the inner segment of human rods extends alongside the outer segment to form a structure hereby termed the "accessory inner segment". While reminiscent of the actin-based microvilli known as "calyceal processes" observed in other species, the accessory inner segment is a unique structure: (1) it contains an extensive microtubule-based cytoskeleton, (2) it extends far alongside the outer segment, (3) its diameter is comparable to that of the outer segment, (4) it contains numerous mitochondria, and (5) it forms electron-dense structures that likely mediate adhesion to the outer segment. Given that the spacing of extrafoveal human photoreceptors is more sparse than in non-primate species, with vast amounts of interphotoreceptor matrix present between cells, the closely apposed accessory inner segment likely provides structural support to the outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.

Investigating the relationship between physical cognitive tasks and a social cognitive task in a wild bird

Despite considerable research into the structure of cognition in non-human animal species, there is still much debate as to whether animal cognition is organised as a series of discrete domains or an overarching general cognitive factor. In humans, the existence of general intelligence is widely accepted, but less work has been undertaken in animal psychometrics to address this question. The relatively few studies on non-primate animal species that do investigate the structure of cognition rarely include tasks assessing social cognition and focus instead on physical cognitive tasks. In this study, we tested 36 wild Western Australian magpies (Gymnorhina tibicen dorsalis) on a battery of three physical (associative learning, spatial memory, and numerical assessment) and one social (observational spatial memory) cognitive task, to investigate if cognition in this species fits a general cognitive factor model, or instead one of separate physical and social cognitive domains. A principal component analysis (PCA) identified two principal components with eigenvalues exceeding 1; a first component onto which all three physical tasks loaded strongly and positively, and a second component onto which only the social task (observational spatial memory) loaded strongly and positively. These findings provide tentative evidence for separate physical and social cognitive domains in this species, and highlight the importance of including tasks assessing both social and physical cognition in cognitive test batteries.

Modulation of prion protein expression through cryptic splice site manipulation

Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP’s sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5’ untranslated region (5’UTR). This exon is homologous to exon 2 in non-primate species, but contains a start codon that would yield an upstream open reading frame (uORF) with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.

A prominent vertical occipital white matter fasciculus unique to primate brains

Vision in humans and other primates enlists parallel processing streams in the dorsal and ventral visual cortex, known to support spatial and object processing, respectively. These streams are bridged, however, by a prominent white matter tract, the vertical occipital fasciculus (VOF), identified in both classical neuroanatomy and recent diffusion-weighted magnetic resonance imaging (dMRI) studies. Understanding the evolution of the VOF may shed light on its origin, function, and role in visually guided behaviors. To this end, we acquired high-resolution dMRI data from the brains of select mammalian species, including anthropoid and strepsirrhine primates, a tree shrew, rodents, and carnivores. In each species, we attempted to delineate the VOF after first locating the optic radiations in the occipital white matter. In all primate species examined, the optic radiation was flanked laterally by a prominent and coherent white matter fasciculus recognizable as the VOF. By contrast, the equivalent analysis applied to four non-primate species from the same superorder as primates (tree shrew, ground squirrel, paca, and rat) failed to reveal white matter tracts in the equivalent location. Clear evidence for a VOF was also absent in two larger carnivore species (ferret and fox). Although we cannot rule out the existence of minor or differently organized homologous fiber pathways in the non-primate species, the results suggest that the VOF has greatly expanded, or possibly emerged, in the primate lineage. This adaptation likely facilitated the evolution of unique visually guided behaviors in primates, with direct impacts on manual object manipulation, social interactions, and arboreal locomotion.

Mesencephalic trigeminal neuron dendritic structures in the macaque.

It is presumed that the unusual central location of mesencephalic trigeminal neurons is a specialization that allows them to receive synaptic input. However, relatively few synaptic terminals were observed on the somata of mesencephalic trigeminal neurons in macaque monkeys via electron microscopy. This leaves the question of dendritic synaptic terminals open. Unlike the pseudounipolar neurons found in the trigeminal ganglion, some mesencephalic trigeminal neurons have been reported to be multipolar cells exhibiting a number of dendritic processes in non-primate species. To examine whether this morphological feature was also present in macaque monkeys, we retrogradely filled these cells with biotinylated dextran amine by injecting it into the trigeminal nerve entry zone. A portion of the mesencephalic trigeminal neurons exhibited short, poorly branched, dendritic processes. They also exhibited very fine, short processes believed to be somatic spines. Thus, primate trigeminal mesencephalic neurons appear to have specializations aimed at increasing the membrane surface area available for synaptic input.

An Overview of Management Considerations for Mongolian Gerbils (Meriones unguiculatus), Cats (Felis catus), and Dogs (Canis familiaris) as Hosts for Brugia Infection.

Lymphatic filariasis is a mosquito-borne parasitic infection affecting an estimated 51.4 million people. Brugia malayi and Brugia pahangi are used in research because common nonprimate research species such as Mongolian gerbils (Meriones unguiculatus), cats (Felis catus), and dogs (Canis familiaris) can maintain the life cycle of these species of filarial nematodes. Although overall care and management of animals infected with Brugia spp. is relatively straightforward, there are some unique challenges and special considerations that must be addressed when managing a research colony infected with these parasites. In this review, we discuss our experience, share insight into biosafety and clinical management, and describe the expected clinical signs associated with Brugia infection in gerbils, cats, and dogs.

Proximity and preening in captive Humboldt penguins

Group-living animals, including penguins, exhibit affiliative behaviors such as grooming (preening) and proximity. Such behaviors in non-primate animals have been less studied than those in primates. Our research focused on 20 identifiable Humboldt penguins in a zoo, analyzing kin relationships and reciprocity in preening and proximity by employing a 5-minute scan sampling method to observe and record individual behavior. Our findings revealed that preening and proximity were more prevalent among mate pairs. However, among non-mate pairs, such behaviors were more commonly observed between siblings and parent-offspring pairs. Notably, the individuals preened on each other simultaneously in all instances. This study highlights the potential influence of kin selection in shaping the affiliative behavior of penguins. Additionally, our findings indicate that penguins gain benefits from mutual preening. This study contributes to our understanding of social behaviors in non-primate species and emphasizes the need for further comparative studies of various animal taxa to elucidate the evolution of sociality.

Evolution of a Human-Specific De Novo Open Reading Frame and Its Linked Transcriptional Silencer.

In the human genome, two short open reading frames (ORFs) separated by a transcriptional silencer and a small intervening sequence stem from the gene SMIM45. The two ORFs show different translational characteristics, and they also show divergent patterns of evolutionary development. The studies presented here describe the evolution of the components of SMIM45. One ORF consists of an ultra-conserved 68 amino acid (aa) sequence, whose origins can be traced beyond the evolutionary age of divergence of the elephant shark, ~462 MYA. The silencer also has ancient origins, but it has a complex and divergent pattern of evolutionary formation, as it overlaps both at the 68 aa ORF and the intervening sequence. The other ORF consists of 107 aa. It develops during primate evolution but is found to originate de novo from an ancestral non-coding genomic region with root origins within the Afrothere clade of placental mammals, whose evolutionary age of divergence is ~99 MYA. The formation of the complete 107 aa ORF during primate evolution is outlined, whereby sequence development is found to occur through biased mutations, with disruptive random mutations that also occur but lead to a dead-end. The 107 aa ORF is of particular significance, as there is evidence to suggest it is a protein that may function in human brain development. Its evolutionary formation presents a view of a human-specific ORF and its linked silencer that were predetermined in non-primate ancestral species. The genomic position of the silencer offers interesting possibilities for the regulation of transcription of the 107 aa ORF. A hypothesis is presented with respect to possible spatiotemporal expression of the 107 aa ORF in embryonic tissues.

Female frogs communicate with males through blinking

Blinking is a behavior unique to animal taxa with motile eyelids, such as most amphibians and reptiles as well as all birds and mammals1. Eyelid movement has physiological functions, such as lubricating the cornea and washing away dust, but its potential signaling functions are not well understood1,2. The use of eyeblinks as a social signal is currently thought to be restricted to some primates, especially humans and their companion animals, but has not been verified in other taxa1,3,4. Here, through field observation and experiments, we demonstrate that female concave-eared torrent frogs (Odorrana tormota), which inhabit noisy streams, use eyeblinks to communicate with males to urge them to initiate amplexus for mating. Our findings reveal that eyeblinks may serve as a social signal in non-primate species.

Novel Tree Shrew Cytochrome P450 2Ds (CYP2D8a and CYP2D8b) Are Functional Drug-Metabolizing Enzymes that Metabolize Bufuralol and Dextromethorphan.

Tree shrews are a nonprimate species used in a range of biomedical studies. Recent genome analysis of tree shrews found that the sequence identities and the numbers of genes of cytochrome P450 (CYP or P450), an important family of drug-metabolizing enzymes, are similar to those of humans. However, tree shrew P450s have not yet been sufficiently identified and analyzed. In this study, novel CYP2D8a and CYP2D8b cDNAs were isolated from tree shrew liver and were characterized, along with human CYP2D6, dog CYP2D15, and pig CYP2D25. The amino acid sequences of these tree shrew CYP2Ds were 75%-78% identical to human CYP2D6, and phylogenetic analysis showed that they were more closely related to human CYP2D6 than rat CYP2Ds, similar to dog and pig CYP2Ds. For tree shrew CYP2D8b, two additional transcripts were isolated that contained different patterns of deletion. The gene and genome structures of CYP2Ds are generally similar in dogs, humans, pigs, and tree shrews. Tree shrew CYP2D8a mRNA was most abundantly expressed in liver, among the tissue types analyzed, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Tree shrew CYP2D8b mRNA was also expressed in liver, but at a level 7.3-fold lower than CYP2D8a mRNA. Liver microsomes and recombinant protein of both tree shrew CYP2Ds metabolized bufuralol and dextromethorphan, selective substrates of human CYP2D6, but the activity level of CYP2D8a greatly exceeded that of CYP2D8b. These results suggest that tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver. SIGNIFICANCE STATEMENT: Novel tree shrew CYP2D8a and CYP2D8b cDNAs were isolated from liver. Their amino acid sequences were 75%-78% identical to human CYP2D6. For CYP2D8b, two additional transcripts contained different patterns of deletion. Tree shrew CYP2D8a mRNA was abundantly expressed in liver, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Recombinant tree shrew CYP2Ds catalyzed the oxidation of bufuralol and dextromethorphan. Tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver.

Frequency selectivity in monkey auditory nerve studied with suprathreshold multicomponent stimuli

Data from non-human primates can help extend observations from non-primate species to humans. Here we report measurements on the auditory nerve of macaque monkeys in the context of a controversial topic important to human hearing. A range of techniques have been used to examine the claim, which is not generally accepted, that human frequency tuning is sharper than traditionally thought, and sharper than in commonly used animal models. Data from single auditory-nerve fibers occupy a pivotal position to examine this claim, but are not available for humans. A previous study reported sharper tuning in auditory-nerve fibers of macaque relative to the cat. A limitation of these and other single-fiber data is that frequency selectivity was measured with tonal threshold-tuning curves, which do not directly assess spectral filtering and whose shape is sharpened by cochlear nonlinearity. Our aim was to measure spectral filtering with wideband suprathreshold stimuli in the macaque auditory nerve. We obtained responses of single nerve fibers of anesthetized macaque monkeys and cats to a suprathreshold, wideband, multicomponent stimulus designed to allow characterization of spectral filtering at any cochlear locus. Quantitatively the differences between the two species are smaller than in previous studies, but consistent with these studies the filters obtained show a trend of sharper tuning in macaque, relative to the cat, for fibers in the basal half of the cochlea. We also examined differences in group delay measured on the phase data near the characteristic frequency versus in the low-frequency tail. The phase data are consistent with the interpretation of sharper frequency tuning in monkey in the basal half of the cochlea. We conclude that use of suprathreshold, wide-band stimuli supports the interpretation of sharper frequency selectivity in macaque nerve fibers relative to the cat, although the difference is less marked than apparent from the assessment with tonal threshold-based data.

On the functional independence of numerical acuity and visual working memory.

Deciding where to direct our vehicle in a crowded parking area or where to line up at an airport gateway relies on our ability to appraise the numerosity of multitudes at a glimpse and react accordingly. Approximating numerosities without actually counting is an ontogenetically and phylogenetically primordial ability, given its presence in human infants shortly after birth, and in primate and non-primate animal species. Prior research in the field suggested that numerosity approximation is a ballistic automatism that has little to do with human cognition as commonly intended. Here, we measured visual working memory capacity using a state-of-the-art change detection task and numerosity approximation using a dot-comparison task, and found a null correlation between these two parametrical domains. By checking the evidential strength of the tested correlation using both classic and Bayesian analytical approaches, as well as the construct validity for working memory capacity and numerosity approximation estimates, we concluded that the present psychophysical evidence was sufficiently strong to support the view that visual working memory and numerosity approximation are likely to rely on functionally independent stages of processing of the human cognitive architecture.

Distinct phenotype of SARS-CoV-2 Omicron BA.1 in human primary cells but no increased host range in cell lines of putative mammalian reservoir species

SARS-CoV-2′s genetic plasticity has led to several variants of concern (VOCs). Here we studied replicative capacity for seven SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, and Omicron BA.1) in primary reconstituted airway epithelia (HAE) and lung-derived cell lines. Furthermore, to investigate the host range of Delta and Omicron compared to ancestral SARS-CoV-2, we assessed replication in 17 cell lines from 11 non-primate mammalian species, including bats, rodents, insectivores and carnivores. Only Omicron's phenotype differed in vitro, with rapid but short replication and efficient production of infectious virus in nasal HAEs, in contrast to other VOCs, but not in lung cell lines. No increased infection efficiency for other species was observed, but Delta and Omicron infection efficiency was increased in A549 cells. Notably replication in A549 and Calu3 cells was lower than in nasal HAE. Our results suggest better adaptation of VOCs towards humans, without an extended host range, and may be relevant to the search for the putative intermediate host and reservoirs prior to the pandemic.

Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol

Cytochromes P450 (CYPs or P450s) are important enzymes for drug metabolism. Tree shrews are non-primate animal species used in various fields of biomedical research, including infection (especially hepatitis viruses), depression, and myopia. A recent tree shrew genome analysis indicated that the sequences and the numbers of P450 genes are similar to those of humans; however, P450s have not been adequately identified and analysed in this species. In this study, a novel CYP2E1 was isolated from tree shrew liver and was characterised in comparison with human, dog, and pig CYP2E1. Tree shrew CYP2E1 and human CYP2E1 showed high amino acid sequence identity (83%) and were closely related in a phylogenetic tree. Gene and genome structures of CYP2E1 were generally similar in humans, dogs, pigs, and tree shrews. Tissue expression patterns showed that tree shrew CYP2E1 mRNA was predominantly expressed in liver, just as for dog and pig CYP2E1 mRNAs. In tree shrews, recombinant CYP2E1 protein and liver microsomes metabolised chlorzoxazone and p-nitrophenol, probe substrates of human CYP2E1, just as they do in dogs and pigs. These results suggest that tree shrew CYP2E1 encodes a functional drug-metabolising enzyme that plays a role in the liver, similar to human CYP2E1.

Von Economo neurons as a specialized neuron class of the human cerebral cortex

By studying human cortical cytoarchitecture, von Economo noticed large spindle-shaped-neurons within layer Vb in the anterior-cingulate and fronto-insular cortex. Those neurons had such extremely elongated stick-like or corkscrew-like soma shape that appeared to him as a pathological alteration. Eventually, he realized that this was a specialized-type of neuron which he described as distinct from the main cortical cell populations, including the commonly found spindle cells. Data from recent studies suggest that specialized-stick-corkscrew-neurons may have first developed in the fronto-insular cortex before the division of hominids and Old World monkeys, and that they have become abundant in the anterior-cingulate cortex only in the hominid line. Golgi analysis found that they have distinctive somato-dendritic morphology with a characteristic very distal position of their axon origin. Many additional studies claimed to find cells similar to the specialized cells described by von Economo in other non-primate species, even in functionally unrelated cortical regions and layers. However, these studies did not provide sufficient evidence that the cells they described are indeed distinct from common spindle-shaped-neurons, and that they truly correspond to the specialized-stick-corkscrew-cells described by von Economo. We believe that present evidence primarily supports the presence of specialized-stick-corkscrew-neurons in hominids, with a seeming increase in their number in humans compared to other primates. The functional significance of such neuronal specialization within specific areas of the human cerebral cortex remains to be elucidated.

Development of Hand and Paw Preferences and Their Association with Other Patterns of Behaviour and Cognition

Hand preference in non-human primates has been studied extensively with the aim of understanding the evolution of hemispheric asymmetry and hand preferences in humans. However, the focus has been on hand preferences expressed in adulthood, with a surprising lack of studies on hand preferences in infants and changes that occur during the development of other, potentially associated, asymmetries in the brain and behaviour. This paper reports on the development of hand preference for grasping food and taking it to the mouth in common marmosets. It considers the development of other types of behaviour, such as head cocking and anogenital licking, that parallel and might influence the development of hand preferences during the first months of life. It then discusses behavioural differences between left- and right-handed adult marmosets, including response to novel stimuli, social behaviour and cognitive bias. The need to study the development of hand preferences together with the development of these other expressions of cognitive function is highlighted. The question to be addressed by empirical studies is whether hand preference is a downstream manifestation of the development of hemispheric differences in sensory processing and cognition, or whether it is instrumental in the development of functional differences between the hemispheres. Comparison is made to paw preference and associated behaviour in non-primate species.

The Impact of Visitors on Non-Primate Species in Zoos: A Quantitative Review.

Visitors are a prominent feature in the lives of zoo animals, and their presence can cause a range of impacts on zoo animals (typically classed as positive, negative or neutral impacts), commonly referred to as the 'visitor effect'. This paper quantitatively collates the literature on the visitor effect in non-primate species, investigates the types of measures used to assess impacts of visitors on animals and considers whether impacts vary across non-primate species in zoos. In total, there were 105 papers which had investigated the impact of zoo visitors on 252 non-primate species/species groups. There has been a steady increase in visitor effect research in zoos since 2012 and this body of work incorporates species from avian (28% study species), reptilian (9%), amphibian (2%), fish (4%) and invertebrate taxa (1%). However, there is still a bias towards mammalian species (56%). The response to visitors varied across taxa. Amphibians responded negatively to visitors more frequently than would be expected by chance (p < 0.05), birds responded neutrally more frequently than would be expected by chance (p < 0.05) and fish responded neutrally and 'unknown' more frequently than would be expected by chance (p < 0.05). This review highlighted a number of animal-based metrics which have been used to assess the impacts of visitors on animals, with measures used varying across taxa. Moving forwards, it is recommended that moving forwards researchers incorporate a suite of measures, incorporating those which are meaningful in terms of being representative of individual animal experiences and animal welfare, collected in a manner which should capture those metrics accurately.

Exogenous and endogenous spatial attention in crows

Attention describes the ability to selectively process a particular aspect of the environment at the expense of others. Despite the significance of selective processing, the types and scopes of attentional mechanisms in nonprimate species remain underexplored. We trained four carrion crows in Posner spatial cueing tasks using two separate protocols where the attention-capturing cues are shown at different times before target onset at either the same or a different location as the impending target. To probe automatic bottom-up, or exogenous, attention, two naïve crows were tested with a cue that had no predictive value concerning the location of the subsequent target. To examine volitional top-down, or endogenous, attention, the other two crows were tested with the previously learned cues that predicted the impending target location. Comparing the performance for valid (cue and target at same location) and invalid (cue and target at opposing locations) cues in the nonpredictive cue condition showed a transient, mild reaction time advantage signifying exogenous attention. In contrast, there was a strong and long-lasting performance advantage for the valid conditions with predictive cues indicating endogenous attention. Together, these results demonstrate that crows possess two different attention mechanisms (exogenous and endogenous). These findings signify that crows possess a substantial attentional capacity and robust cognitive control over attention allocation.