
==== Front
Genes Brain Behav
Genes Brain Behav
10.1111/(ISSN)1601-183X
GBB
Genes, Brain, and Behavior
1601-1848
1601-183X
Blackwell Publishing Ltd Oxford, UK

10.1111/gbb.12907
GBB12907
Original Article
Original Article
Divergent neural nodes are species‐ and hormone‐dependent in the brood parasitic brain
Duque et al.
Duque Fernanda G. https://orcid.org/0000-0002-1446-8929
1 2 fgduque@ilstu.edu

Azam Asma 1
Kaur Amanpreet 1
Pao Rachel 1
Lynch Kathleen S. 1
1 Biology Department Hofstra University Hempstead New York USA
2 School of Biological Sciences Illinois State University Normal Illinois USA
* Correspondence
Fernanda G. Duque, School of Biological Sciences, Illinois State University, Normal, IL 61790, USA.
Email: fgduque@ilstu.edu

08 9 2024
10 2024
23 5 10.1111/gbb.v23.5 e1290702 7 2024
17 10 2023
04 7 2024
© 2024 The Author(s). Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Abstract

Avian brood parasitism is an evolutionarily derived behavior for which the neurobiological mechanisms are mostly unexplored. We aimed to identify brain regions that have diverged in the brood‐parasitic brain using relative transcript abundance of social neuropeptides and receptors. We compared behavioral responses and transcript abundance in three brain regions in the brown‐headed cowbird (BHCO), a brood parasite, and a closely related parental species, the red‐winged blackbird (RWBL). Females of both species were treated with mesotocin (MT; avian homolog of oxytocin) or saline prior to exposure to nest stimuli. Results reveal that MT promotes approach toward nests with eggs rather than nests with begging nestlings in both species. We also examined relative transcript abundance of the five social neuropeptides and receptors in the brain regions examined: preoptic area (POA), paraventricular nucleus (PVN) and bed nucleus of the stria terminalis (BST). We found that MT‐treated cowbirds but not blackbirds exhibited lower transcript abundance for two receptors, corticotropin‐releasing factor 2 (CRFR2) and prolactin receptor (PRLR) in BST. Additionally, MT‐treated cowbirds had higher PRLR in POA, comparable to those found in blackbirds, regardless of treatment. No other transcripts of interest exhibited significant differences as a result of MT treatment, but we found a significant effect of species in the three regions. Together, these results indicate that POA, PVN, and BST represent neural nodes that have diverged in avian brood parasites and may serve as neural substrates of brood‐parasitic behavior.

Mesotocin (MT, homologous to oxytocin in mammals) enhances natural preferences for egg‐related stimuli in brood‐parasitic and parental females alike. In the brain, MT treatment was associated with changes in gene expression in the bed nucleus of the stria terminalis and the preoptic area in brood parasitic females.

bed nucleus stria terminalis
paraventricular nucleus
parental care
preoptic area
social behavior network
social neuropeptides
National Science Foundation 10.13039/100000001 1949188 source-schema-version-number2.0
cover-dateOctober 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:09.09.2024
Duque FG , Azam A , Kaur A , Pao R , Lynch KS . Divergent neural nodes are species‐ and hormone‐dependent in the brood parasitic brain. Genes, Brain and Behavior. 2024;23 (5 ):e12907. doi:10.1111/gbb.12907

Asma Azam, Amanpreet Kaur, and Rachel Pao contributed equally to this study.
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pmc1 INTRODUCTION

Avian brood parasites are a classic example of a species that displays a behavior that is evolutionarily derived rather than ancestral. Providing parental care is the ancestral state in birds and therefore, brood parasites that rely on parental care from other species rather than providing it themselves represent a major evolutionary transition that has occurred in roughly 1% of bird species. 1 The loss of parental care has occurred seven independent times across five families of birds. 2 , 3 , 4 While there is a long history of research into the ultimate explanations for the loss of parental care, there are few investigations into the proximate mechanisms of this behavior. Understanding the neurobiological basis for this behavior, however, requires a fundamental understanding of the molecular and neural pathways that may have diverged in brood parasites so that we can begin to identify where and what to look for in the brain of parasitic species. The present study is aimed at identifying neural pathways that exhibit divergence in the brood‐parasitic brain so that we begin unraveling the mechanistic basis of brood‐parasitic behavior.

Parental care enhances offspring survival and the fitness of individuals that successfully raise their young to independence. The suite of behaviors that comprise parental care may represent the most ancient caregiving system and therefore, the neural networks involved in parental care may serve as foundational circuitry for other forms of social bonds to evolve. 5 If this is the case, conserved neural networks underlying parental care may have been co‐opted and subsequently shaped by selection to produce a variety of bonding behaviors including pair‐bonding in monogamous mating systems. 5 These conserved neural pathways form a network within the limbic forebrain and midbrain in amniotes, including mammals and birds, broadly known as the “social behavior neural network” (SBN). 6 The neural pathways involved in parental care are thus well conserved and may have served as substrates for evolution, modifying the role of these regions in the regulation of social behaviors 6 , 7 and producing variation in the quality and quantity of parental care across vertebrates.

Brain nuclei within the SBN express high densities of sex steroids, catecholamines, and their receptors, and they are essential for the expression of a wide range of social behaviors including reproduction, social recognition and learning, aggression, and parental care. 5 , 6 , 7 , 8 The social‐regulating capacity of these regions is due, in large part, to specific cell types that contain nonapeptides that regulate social behaviors (hereafter referred to as “social neuropeptides”) and their receptors. Social neuropeptides such as oxytocin (OXT; and its non‐mammalian homolog mesotocin (MT) 9 ) act as mediators of several social behaviors across various vertebrates 6 , 10 , 11 , 12 , 13 , 14 , 15 , 16 including caregiving and pair‐bonding. 5 , 17 Social neuropeptides such as OXT are mostly produced in hypothalamic regions such as the preoptic area (POA), paraventricular nucleus (PVN) and the supraoptic nucleus (SON), which send widespread neuropeptide‐containing projections across the brain, 18 and extrahypothalamic OXT projections are mostly derived from the PVN. 19 , 20 , 21 In addition to these hypothalamic regions, the bed nucleus of the stria terminalis (BST) is also rich in social neuropeptides and contain cells that project to other basal forebrain regions, including those involved in regulation of social behavior such as the lateral septum. 22 The BST is a highly plastic, steroid‐dependent system that is known to modulate various affiliative, caregiving, and anxiety‐like behaviors across vertebrates. 16 , 23 , 24 Moreover, many of these regions also encode positively and negatively valanced social stimuli. 22 , 25 , 26 , 27 Valence refers to the positive or negative value assigned to a particular stimulus or signal. For example, a predator call may have a negative valence because it is a threat, while a courtship song during the breeding season may have a positive valence if the female is receptive. In both cases, the signals are relevant to the female, but the assessment of each varies. Differences in valence encoding of some social signals such as begging calls may be a key neural component involved in brood‐parasitic behavior. Furthermore, these regions are within the well‐defined SBN that regulates social decision‐making and contains social neuropeptides as well as their receptors. 6 , 22 , 28

The brown‐headed cowbird (Molothrus ater; BHCO) is a ubiquitous brood parasite across North America and is related to the red‐winged blackbird (Agelaius phoeniceus; RWBL) a parental species that also serves as a preferred host for the brown‐headed cowbird in many parts of its range. 29 , 30 Because parental care is an ancestral trait in birds, 31 the loss of parental behaviors in cowbirds likely occurred in recent evolutionary times between 6 and 2.5 Mya 32 , 33 , 34 after the divergence from blackbirds (Figure 1A). Here, we aimed to understand the neurobiological basis for this stark shift in parental behavior by comparing the behavioral and neural responses of these two related species (Family Icteridae) to biologically relevant stimuli.

FIGURE 1 Experimental design. (A) Blackbird phylogeny showing in red the Agelaius genus that includes the red‐winged blackbird, and in brown, brood‐parasitic cowbirds in the genus Molothrus, including the brown‐headed cowbird. Phylogeny based on Barker, et al. 2013; Powell, et al. 2014. (B) Schematic of the behavioral cage depicting a brown‐headed cowbird female in the neutral zone. A red dashed rectangle shows the ‘approach’ area approximately 10 cm around each nest. (C) Experimental timeline for the behavioral test and sample collection. (D) Schematic of a representative coronal section showing where punches were collected from the Bed nucleus of the stria terminalis (BST), preoptic area (POA), and paraventricular nucleus (PVN). Brain schematic redrawn from Lynch, et al. (2020).

We compared preference behaviors toward parental versus reproductive stimuli followed by measurements of transcript abundance in our target brain regions across mesotocin (MT)‐treated and ‐untreated females of both species. We hypothesize that regions in the social behavior neural network have diverged in avian brood parasites compared with parental species, which may have facilitated or was a consequence of the transition to the behavioral phenotypes typical of brood parasitic species. Thus, we predict the following: (1) red‐winged blackbirds will exhibit greater preference behaviors toward nestling‐related stimuli compared with brown‐headed cowbirds, whereas cowbirds will prefer to approach eggs in the nest; (2) MT will enhance this difference for preference of nestling‐related stimuli between the red‐winged blackbird and brown‐headed cowbirds and (3) these species‐ and MT‐dependent behavioral differences will be reflected in transcript abundance of social neuropeptides and their receptors within key brain regions (i.e., BST, POA and PVN). The latter prediction will allow us to identify neural nodes that have diverged in the cowbird and the role of MT in activating these differences. Moreover, determining whether MT modifies the transcript abundance of social neuropeptides in a species‐dependent manner will suggest that transcript differences in the brain between these species can be activated by hormones, thereby suggesting that activational effects of hormones may be a part of the regulatory mechanisms that promote neural differences between parental and non‐parental blackbirds. Here, we assessed expression profiles of five transcripts across the POA, PVN, and BST, which are involved in regulating pair‐bonding and caregiving social behaviors. 6 , 22 , 23 , 25 The transcripts selected for examination in this study are: (1) peptide hormones and receptors that are known to be involved in the regulation of pair‐bonding behavior including parental care, (2) known to be expressed in one or more of our target brain regions, or (3) identified in our previous Next Generation Sequencing (NGS) study as differentially expressed in the POA between brown‐headed cowbird and red‐winged blackbird. 35 , 36 The selected transcripts include prolactin receptor (PRLR), corticotropin‐releasing factor receptor type 2 (CRFR2), galanin (GAL), arginine vasotocin (AVT), and mesotocin (MT).

2 MATERIALS AND METHODS

2.1 Animal collection and care

Brown‐headed cowbird females were collected via drop‐in traps in Queens, NY from July to August 2021, and red‐winged blackbird females were collected via walk‐in traps and mist nests from May to June 2022 in Lisbon, ND. All red‐winged blackbird females were building nests or incubating eggs at the time of collection. The breeding season in brown‐headed cowbirds extends from April to August, while in red‐winged blackbirds, it starts in April but ends around mid‐July. 37 , 38 While the behavior of the red‐winged blackbird changes throughout the breeding season due to the different demands of egg laying and offspring rearing, the brown‐headed cowbird lays eggs throughout the entire breeding season.

All birds were collected during the breeding season. Females of both species were provided with food and water ad libitum for a week before conducting behavioral experiments. All scientific collections and experimental procedures were approved by Hofstra University's IACUC as well as permitted by federal (MB96705A) and state agencies (OLN05722779 ND; 1181 NY).

2.2 Behavioral assay and mesotocin (MT) treatment

Brown‐headed cowbirds do not exhibit any form of parental care whereas red‐winged blackbird females exhibit a full suite of caregiving behaviors including building nests, incubating eggs, and provisioning offspring until independence. Thus, our behavioral experiments aimed to compare parental‐like behaviors in these two species; one of which does not naturally provide any parental care. Therefore, we measured the female's preference to approach various biologically relevant stimuli that typically induce caretaking in birds, including eggs, nestling models, and begging sounds. While eggs do not induce incubation in cowbirds, they are a biologically relevant stimulus because cowbirds tend to parasitize nest containing eggs. 39

The preference tests in the behavioral cage consisted of three chambers: a neutral zone in the middle and two side chambers containing the experimental stimuli (Figure 1B). Each side chamber contained stimuli for the birds to approach, which will be referred to hereafter as the nestling stimulus or the egg stimulus. The nestling stimulus included a nest containing a model nestling covered in commercially available down feathers. The beak and gape of the nestling model, however, was taken from an actual brown‐headed cowbird nestling, dried, and painted in yellow in the flanges and bright red in the gape. Inside the beak was painted red to mimic the gape of a begging nestling because the gape color is one of the relevant stimuli for provisioning parents. 40 , 41 The gape of the nestling model was fixed in a wide‐open position to mimic a begging nestling, and it was paired with begging calls broadcast from the nest. The egg stimulus consisted of a similar nest with model eggs paired with dove coos broadcast from the nest. Dove coos are heterospecific calls that do not resemble any conspecific vocalizations for cowbirds or red‐winged blackbirds. Moreover, mourning dove nests are rarely parasitized by cowbirds because doves are inappropriate hosts. 42 , 43 Likewise, red‐winged blackbirds are not expected to show interest in a nest with eggs that is associated with heterospecific sounds. Thus, dove coos serve as a positive control for the presence of sound.

Sound was played back using Auvio Mono Plus Bluetooth speakers (Radio Shack, Forth Worth, TX, USA). Recordings from nests that included a brown‐headed cowbird nestling were used to generate an acoustic stimulus of nestling begging calls and dove coos were used to generate a control stimulus. Both stimuli were generated from publicly available recordings obtained from Xeno‐Canto (www.xeno-canto.org). Audacity v. 3.1.3 44 was used to generate these stimuli from the sound recordings. We generated three recordings, one to be played during the initial acclimation phase, and two recordings for the behavioral trials, allowing us to avoid habituation to the sound stimuli being presented. Recordings of begging calls or dove coos were normalized to 70 dB and assembled with random silence intervals to build 40‐s bouts of each stimulus. In the first behavioral trial, the final acoustic stimuli consisted of a 15‐min recording composed of six bouts of begging calls and six bouts of dove coos presented in random order and interleaved with silence intervals of random duration. For the second trial, a second recording was created following the same parameters; however, the first sound presented to the bird was alternated with respect to the recording used in the previous trial. The recording played during the initial acclimation phase followed the same parameters with additional 5 min of silence interleaved with the bouts of sound to reach a final time of 20 min. Sound pressure levels were measured using a mobile sound meter application, Sound Meter v. 1.7.18 (Smart Tools, South Korea), at the beginning of each experimental day to confirm that the sound levels were consistent throughout the experiment. Therefore, all acoustic stimuli were matched in amplitude and duration.

Mesotocin solution was prepared using Ile8‐Oxytocin trifluoroacetate (Cat. No. 4030888, Bachem, Torrance, CA, USA) in saline solution with a concentration of 1 ng/uL, comparable to dosage employed in studies with rodents 45 , 46 , 47 ; 50 uL aliquots were stored at −20°C and thawed at 4°C about 10 min before administration. Procedures for intranasal oxytocin administration were modified from another study in birds. 48 Briefly, females in the experimental group received 50 uL of MT solution administered intranasally 30 min before the start of the behavioral test (BHCO, n = 12; RWBL, n = 9), whereas control females received 50 uL of saline solution (BHCO, n = 13; RWBL, n = 6).

At the start of each behavioral trial, females were exposed to the auditory stimuli for 20 min while in the neutral zone (Figure 1B). Immediately after, side doors were opened allowing the female to explore either side chamber containing the nestling stimulus or the egg stimulus for 15 min. After the first trial, stimuli switched sides and the female was tested for additional 15 min to avoid measurements of side bias rather than stimuli preference. In total, behavior was observed for 30 min in each female (see Figure 1C for the experimental timeline). All behavioral assays were recorded with a Hero 4 camera (GOPRO Inc., San Mateo, CA, USA) in front of the behavioral chamber. Variables measured from behavioral observations include (1) total time females spent on the experimental side with the nestling‐related stimuli versus time spent on the positive control side with the egg paired with a heterospecific call and, (2) total number of approaches (i.e., female perching within 10 cm of the nest or on the nest).

2.3 Tissue sample collection and RT‐qPCR

Immediately following behavioral assays, females were sacrificed via rapid decapitation. Brains were flash frozen on dry ice and stored at −80°C until sectioning. Brain tissue was sectioned coronally at 200 μm, and bilateral punches were collected using a 1.0 mm brain punch tool (Cat. No. 15112‐100, Ted Pella Inc., Redding, CA, USA) from three brain regions following landmarks described in references 36, 49, 50, 51, 52. Briefly, sections of the BST were collected starting at the most ventral part of the lateral ventricle until the anterior commissure was visible. Sections of the POA were collected starting at the area between the septomesencephalic tract until the anterior commissure was visible. Finally, PVN sections were collected from regions surrounding the dorsal end of the third ventricle (Figure 1D). RNA was isolated via Trizol reagent (Cat. No. AS1270, Promega, Madison, WI, USA) using manufacturers specification followed by cleanup using TURBO DNA‐free kit (Cat. No. AM1907, Thermo Fisher Scientific, Waltham, MA, USA). Because we obtained a Maxwell 16 MDx instrument (Promega, Madison, WI, USA) to handle the large number of RNA isolations, RNA was also isolated from the remaining tissue by following manufacturer specifications for the Maxwell 16 machine. There were no differences in the quality of RNA isolated using these two techniques as measured by a BioTek Synergy HTX multi‐mode reader (Agilent Technologies, Inc., Santa Clara, CA, USA). Reverse transcription into cDNA was completed using SuperScript First Strand synthesis for Real‐Time Polymerase Chain Reaction (RT‐PCR) (Cat. No. 11904‐018, Thermo Fisher Scientific, Waltham, MA, USA); cDNA samples were stored in −20°C until the RT‐qPCR procedure.

Previous studies using NextGen sequencing in the POA of brown‐headed cowbirds and red‐winged blackbirds revealed thousands of transcripts that were not differentially expressed between these two species. 35 This evidence allowed us to focus our efforts on investigating differential expression of transcripts with a known role in modulating social behavior, especially parental care. Prior information revealing differential transcript abundance between these two species occurs in only specific transcripts rather than widely in the transcriptome. RT‐qPCR was conducted as described in references 35, 36, 53. Briefly, our procedures use Power SYBR green PCR Master Mix by Applied Biosystems (Cat. No. 4367659, Thermo Fisher Scientific, Waltham, MA, USA), and CFX Connect RT‐PCR Detection System (Bio‐Rad Laboratories, Hercules, CA, USA). Supplementary Table 1 shows the list of transcripts and primers used in the RT‐qPCR procedure. Samples were run in triplicates, and transcript abundance was estimated based on the average across triplicates. Relative quantities were then normalized based on total starting cDNA concentration per sample. We used a Quant‐it Ribogreen RNA assay kit (Cat. No. R11490, Thermo Fisher Scientific, Waltham, MA, USA), which is equally effective at measuring single stranded RNA and cDNA. 36 , 53 Normalized values were then log transformed for statistical analysis. Final sample sizes for each transcript depended on quantities of cDNA remaining and detection threshold during RT‐qPCR (Supplementary Table 2).

2.4 Statistical analysis

All statistical analyses were performed using the R software version 4.2.2 54 including base functions; graphs were generated using ggplot2. 55

2.4.1 Behavioral analysis

To measure the effect of intranasal administration of MT on preference response to nestling stimulus versus eggs stimulus, we first measured whether there were differences in the total time female brown‐headed cowbirds and red‐winged blackbirds spent outside of the neutral zone. Because the data followed a gamma distribution, we fitted a generalized linear model (GLM) with gamma distribution using the “glm” function in R. 54 To account for a potential effect of the time of the breeding season, we included this variable in our model. However, we did not find a significant effect of time of the breeding season in the behavioral measurements we analyzed (Supplementary Table 3). Thus, we removed this variable from the model to increase our statistical power to detect other significant effects. For total time outside of the neutral zone, we did not find a significant effect of MT treatment (t 39 = 0.905, p = 0.37), but there was a significant effect of species (t 39 = 2.421, p = 0.0207) showing that red‐winged blackbird females spent more time outside of the neutral zone compared with cowbird females (RWBL, mean ± standard error of the mean (SEM) = 1666 ± 73.83; BHCO, mean ± SEM = 1134.3 ± 113.92; Supplementary Figure S1A,B). Therefore, our preference measures (i.e., time spent on each stimulus side and number of approaches to each stimulus) were normalized by the total time females spent outside the neutral zone and total number of approaches to account for species differences in total activity outside the neutral zone. Because we were working with bounded data (range −1 to 1), we transformed the data to proportions and fitted a beta regression 56 using the betareg package. 57 Finally, we evaluated pairwise comparisons using estimated marginal means and adjusted for multiple comparisons using the false discovery rate (FDR) post hoc method with the lsmeans package. 58

2.4.2 Relative transcript abundance across neural nodes

We first measured differences in relative transcript abundance across species, treatment, brain region, and time of the breeding season. For this analysis, we fitted a linear mixed model (LMM) for each transcript of interest. We did not find a significant effect of time of the breeding season in the relative abundance of any of the transcripts we analyzed (Supplementary Table 4). Thus, we removed this variable from the model to increase our statistical power to detect other significant effects. All LMMs were fitted and the significance of the factors was tested using the lmerTest package. 59 In the case of transcripts for which we did not find a significant effect of MT treatment or a biologically relevant interaction, we removed this variable from the analysis and fitted the models only using species and brain region as independent variables. Pairwise comparisons were assessed using estimated marginal means and adjusted for multiple comparisons with Tukey's posthoc analysis using the emmeans package. 60 For transcripts in which we found a significant effect of MT treatment, we kept MT treatment in the model. We then evaluated pairwise comparisons using estimated marginal means and corrected for multiple comparisons using false discovery rate (FDR) in the lsmeans package 58 to account for the substantial increase in the number of pairwise comparisons.

2.4.3 Co‐expression of transcripts of interest across neural nodes

To identify patterns of co‐expression of neuropeptides and receptors across brain regions, we built correlation matrices across transcripts in the three brain regions in both species using the “cor” function in R 54 and the reshape2 package 61 to understand the magnitude of the co‐expression. Then, using the rstatix package, 62 we evaluated the significance of those relationships.

3 RESULTS

3.1 Behavioral results

With respect to the difference in total time spent near nestling stimulus versus egg stimulus, we found a significant effect of MT treatment (Beta regression, Z (5) = −4.057, p < 0.001) but not an effect of species or a significant interaction between these variables (Supplementary Table 5). This result indicates that MT‐treated females in both species spent significantly more time on the side of the cage with the egg stimulus compared with side with the nestling stimulus (mean ± SEM, control: 0.0231 ± 0.113; MT: −0.479 ± 0.089; Figure 2A). With respect to the number of approaches, we found a significant effect of species (Beta regression, Z (5) = −2.708, p = 0.007), MT treatment (Beta regression, Z (5) = −2.761, p = 0.006), and a significant interaction (Beta regression, Z (5) = 2.326, p = 0.02) (Figure 1B, Supplementary Table 5). Pairwise comparisons showed significant differences between saline‐ and MT‐treated cowbirds as well as between cowbird controls and red‐winged blackbirds regardless of treatment (Figure 2B, Supplementary Table 5 for statistics on pairwise comparisons). These results indicate that MT‐treated brown‐headed cowbird females and red‐winged females perched significantly more times near or on the nest with eggs compared with saline‐treated brown‐headed cowbird females.

FIGURE 2 Female approach to nestling stimulus versus egg stimulus. (A) Bar plots showing the proportion of time that females spent in the chamber with each stimulus: Nestling stimulus versus egg stimulus. All data are normalized by the total time females spent outside of the neutral zone. (B) Bar plot showing the proportion of female approaches to the nest with the nestling stimulus compared with the nest with the egg stimulus. All data are normalized by the total number of approaches to either stimulus. Dashed lines in A and B indicate a threshold for female preference for the nestling stimulus (above line, upward arrow in the right) or for the egg stimulus (below line, downward arrow in the right). Control females (saline; BHCO, n = 13; RWBL, n = 6); MT‐treated females (BHCO, n = 12; RWBL, n = 9). BHCO, brown‐headed cowbird; MT, mesotocin; RWLB, red‐winged blackbird. Significant differences across groups are shown by different letters.

3.2 Divergence in neural nodes: Relative transcript abundance across species, treatment, and brain region

When assessing the effects of MT in the abundance of neuropeptide receptors, we found a significant interactions of MT treatment with species and brain region in addition to a significant species effect in the transcript abundance of both candidate genes, CRFR2 and PRLR (Figure 3, Table 1 for main effects, Supplementary Tables 6 and 7 for pairwise comparisons). Regarding CRFR2, results showed that transcript abundance is significantly higher in red‐winged blackbirds than in the cowbirds regardless of treatment. In addition, it was revealed that MT‐treated cowbirds exhibited lower levels of CRFR2 in BST compared with saline‐treated cowbirds (Figure 3). This decrease in transcript abundance was not found in the other brain regions across treatment groups in cowbirds or red‐winged blackbirds.

FIGURE 3 Relative transcript abundance across brain regions. Plots show mean (dot) and standard error of the mean (SEM, bars). Different letters show significant differences in transcript abundance; color red shows a pairwise comparison approaching significance. AVT, arginine vasotocin; BHCO, brown‐headed cowbird; BST, bed nucleus of the stria terminalis; CRFR, corticotropin‐releasing factor receptor; GAL, galanin; MT, mesotocin; POA, preoptic area; PRLR, prolactin receptor; PVN, paraventricular nucleus; RWBL, red‐winged blackbird. (See Table 1 for statistics on linear mixed models and Supplementary Table 6 for pairwise comparisons).

TABLE 1 Statistical tests, main effects, and interactions for five transcripts across three brain regions: Preoptic area (POA), paraventricular nucleus (PVN), and bed nucleus of the stria terminalis (BST). Comparisons were conducted between BHCO and RWBL.

Transcript	Parameter	F statistic	(DFn, DFd)	p	
CRFR	Species	67.62	(1, 18.6)	<0.001	
Treatment	0.194	(1, 18.6)	0.665	
Brain Region	0.038	(2, 30.3)	0.962	
Species*Treatment	0.069	(1, 18.6)	0.796	
Species*Region	0.221	(2, 41)	0.803	
Treatment*Region	8.02	(2, 30.3)	0.002	
Species*Treatment*Region	3.99	(2, 30.3)	0.029	
PRLR	Species	9.919	(1, 19.4)	0.005	
Treatment	0.001	(1, 19.4)	0.971	
Brain Region	0.593	(2, 36.7)	0.558	
Species*Treatment	0.004	(1, 19.4)	0.95	
Species*Region	0.769	(2, 36.7)	0.471	
Treatment*Region	5.098	(2, 36.7)	0.011	
Species*Treatment*Region	6.417	(2, 36.7)	0.004	
AVT	Species	202.276	(1, 27.9)	<0.001	
Brain Region	15.141	(2, 46.4)	<0.001	
Species*Region		(2, 46.4)	<0.001	
GAL	Species	87.616	(1, 26.3)	<0.001	
Brain Region	29.259	(2, 47.2)	<0.001	
Species*Region	0.1265	(2, 47.2)	0.8815	
MT	Species	11.659	(1, 21.3)	0.0026	
Brain Region	117.096	(2, 43.5)	<0.001	
Species*Region	27.27	(2, 43.5)	<0.001	

When assessing PRLR transcript abundance, results revealed that in saline‐treated cowbirds, PRLR levels were significantly lower than in red‐winged blackbirds only in POA whereas BST and PVN exhibited comparable PRLR abundance levels in both species (Figure 3, Table 1 for main effects, and Supplementary Table 7 for pairwise comparisons). Interestingly, PRLR transcript abundance in the POA of MT‐treated cowbirds no longer exhibited a significant difference compared with red‐winged blackbirds. There was a significant decrease of PRLR transcript levels in the BST of MT‐treated cowbirds compared with saline‐treated cowbirds (Figure 3, Supplementary Table 7). Finally, PRLR abundance in the PVN did not exhibit any significant difference across treatment groups or between species.

We found no significant effect of MT treatment in transcript abundance of AVT, GAL, and MT. Thus, we removed MT treatment as a variable from the models and focused on investigating the role of species and brain region in relative transcript abundance (see Supplementary Table 8).

Regarding relative AVT transcript abundance, we found significant effects of species, brain region, and a significant interaction (Table 1 for main effect statistics). Overall, red‐winged blackbird females exhibited higher AVT transcript abundance in the three brain regions compared with brown‐headed cowbirds. There were also differences across brain regions within each species. POA had significantly higher levels of AVT transcript in red‐winged blackbirds compared with BST and PVN. In contrast, brown‐headed cowbird females exhibited higher AVT transcript abundance in PVN relative to BST but not to POA (Figure 3, Supplementary Table 9 for pairwise comparisons).

With respect to relative GAL transcript abundance, we found significant main effects of species and brain region, but there was no interaction between the two variables (Table 1 for statistics on main effects). BST, POA, and PVN in red‐winged blackbirds exhibited higher transcript levels of GAL compared with those regions in brown‐headed cowbirds. There were also differences in transcript abundance across regions that followed the same pattern in both species, so that POA and PVN had higher AVT transcript abundance compared with BST (Figure 3, Supplementary Table 9 for pairwise comparisons).

With respect to relative MT transcript abundance across brain regions in brown‐headed cowbird and red‐winged blackbird females, we found significant effects of species, brain region, and a significant interaction between species and brain region (see Table 1 for main effect statistics). The PVN exhibited significantly higher MT transcript abundance in female brown‐headed cowbirds than in red‐winged blackbirds. In addition, we found that POA and PVN had higher MT transcript abundance than BST in female brown‐headed cowbirds whereas in red‐winged blackbirds, POA showed higher levels of MT transcript than BST and PVN (Figure 3, Supplementary Table 9 for pairwise comparisons).

3.3 Divergence in neural nodes: Co‐expression of transcripts across brain regions

Correlation matrices revealed that the relative abundance of transcripts was significantly correlated in each brain region. The patterns of co‐expression across regions varied in each species, but all significant correlations exhibited high correlation factors, R 2 > 0.6 (Figure 4).

FIGURE 4 Intra‐regional transcript co‐expression. Pairwise correlations between transcripts of interest across brain regions in (A) brown‐headed cowbird females and (B) red‐winged blackbird females. Coefficients are shown for each comparison following a color scale, and numbers inside the matrix represent significant p‐values of each correlation coefficient at the 0.05 level whereas red values show a trend approaching significance. AVT, arginine vasotocin; BST, bed nucleus of the stria terminalis; CRFR, corticotropin‐releasing factor receptor; GAL, galanin; MT, mesotocin; POA, preoptic area; PRLR, prolactin receptor; PVN, paraventricular nucleus.

In BST, we found that AVT was significantly correlated with PRLR and CRFR2 in brown‐headed cowbirds (p < 0.05, Figure 4A), while showing a trend that did not reach significance (p = 0.09) in co‐expression with GAL. In red‐winged blackbirds, AVT was correlated with GAL and CRFR2 (p < 0.05) but not with PRLR (p = 0.08) (Figure 4B). In contrast, PRLR was significantly co‐expressed with CRFR2 in both species, and GAL was also correlated with PRLR and CRFR2 transcripts in both species (Figure 4A,B).

In POA, all transcripts were significantly correlated with one another in the brown‐headed cowbirds (Figure 4A), except for AVT and GAL. In brown‐headed cowbirds, the co‐expression of PRLR and CRFR2 showed the highest correlation value in this analysis (R 2 = 0.98). Meanwhile in red‐winged blackbirds, PRLR was correlated with CRFR2, AVT, and GAL (p < 0.05 for all comparisons, Figure 4B), but not with MT (p = 0.06). GAL was also significantly correlated with MT and AVT, which were correlated with each other (p < 0.05 for all comparisons, Figure 4B).

Finally, we found that in PVN, all transcripts were significantly correlated with one another, exhibiting high correlation factors (R 2 > 0.6) in brown‐headed cowbirds (p < 0.05 for all comparisons, Figure 4A). AVT AND CRFR2 were the exception, showing a trend that did not reach the significance threshold (p = 0.07, Figure 4A). In contrast in red‐winged blackbirds, co‐expression patterns in PVN showed that MT was significantly correlated with AVT only, and PRLR was correlated with GAL and CRFR2, which were also correlated with each other (p < 0.05, Figure 4B).

4 DISCUSSION

We predicted that there would be a species difference in preference for nestling‐related stimuli that would be enhanced after treatment with MT. Specifically, we predicted red‐winged blackbirds would exhibit greater approach behavior toward nestlings, whereas cowbirds would exhibit a greater approach toward eggs, and these differences would become more pronounced after MT treatment. However, our results revealed that intranasal MT administration was associated with a preference for nests with eggs in both species. Both cowbirds and red‐winged blackbirds exhibited a significant increase in the amount of time spent in proximity to a nest containing eggs with a heterospecific song broadcast from the nest (Figure 2) as opposed to begging nestling sounds with a nestling model. While this does not support our prediction, this result is consistent with reports showing that MT in the brain of female Thai chickens increases gradually as they enter egg laying and peak in incubating and nestling stages. 63 However, behavioral effects of MT vary widely and tend to be species‐specific. 64 Studies in pinyon jays, a highly social corvid, and vampire bats reveal increased caregiving behaviors including food sharing and allogrooming with high doses of intranasal MT. 48 , 65 Here, we show that both species exhibit a preference toward nests with eggs when treated with MT, albeit this behavior likely serves different functions in each species. In female red‐winged blackbirds, MT may promote maternal behaviors such as incubation toward eggs as it does in other parental species. 63 The female in our study were collected during nest‐building and incubation stages of the breeding season, which may explain the preference for eggs over begging calls with a nestling model. In cowbirds, however, there is no nest building or incubation stage during the breeding season; therefore, MT may promote a suite of parasitism‐related behaviors such as mafia behaviors 66 and nest searching as cowbirds preferentially parasitize nests with eggs. 39 While this conclusion needs much additional exploration, it is partially supported by repeated observations during behavioral trials in which some female cowbirds approached nests with eggs to aggressively peck at them and/or throw them to the ground; a typical behavior of a cowbird that is laying eggs in a host nest 67 , 68 , 69 (Supplementary Video 1). In contrast, behaviors aimed at destroying eggs did not occur in red‐winged blackbirds. However, as described above, we did not predict that MT would promote preference toward eggs in both species and therefore, the placement of our camera did not allow us to quantify in fine detail the interactions of female red‐wing blackbirds with eggs, which were not as obvious as those of cowbirds. Therefore, our behavioral paradigm requires greater resolution to characterize differences in behavior toward nest contents that may be promoted by MT or other hormones associated with parental behavior. However, in a previously published study from our lab, results of an approach/avoidance behavioral test indicated that cowbirds exhibit avoidance of begging nestling stimuli, 36 similar to patterns observed in this study.

When assessing transcript abundance in the three brain regions, there was a species difference and a significant effect of MT treatment on CRFR and PRLR abundance in the BST. Transcript abundance of CRFR and PRLR decreased in BST of MT‐treated cowbirds compared with saline‐treated cowbirds, whereas this difference did not occur in red‐winged blackbirds (Figure 3). It is not entirely clear why MT treatment would result in a decrease of the abundance of peptide receptors in the BST; however, nonapeptides (i.e., AVT and MT) in the BST regulate social behaviors in songbirds including flocking, 70 aggression, 71 pair bonding 72 and parental care. 23 Thus, if MT treatment decreases abundance of these two receptors, it may also alter receptor abundance for other key peptides in the BST, including nonapeptide receptors. This decline in socially related receptor abundance like PRLR may mediate a change in social behaviors like parental care. While the exact role of CRFR2 in regulating parental behavior is still unclear, there is evidence suggesting that the urocortin peptides that activate this receptor are involved in modulating parental behaviors. 73 , 74 , 75 , 76 Furthermore, these results identify the BST as a neural node in which transcript abundance is different from that of a related parental blackbird and receptor abundance is modulated by changes in hormones, specifically MT. This finding supports the prediction that key brain regions not only exhibit species differences between parental and non‐parental blackbirds, but at least some are differentially modulated by hormones.

We also found a trend showing that MT treatment increased PRLR transcript abundance in the POA of brown‐headed cowbirds. While saline‐treated cowbirds exhibited lower PRLR levels in POA compared with red‐winged blackbirds, which is consistent with previous results, 35 , 36 MT‐treated cowbirds had PRLR transcript levels similar to those found in the POA of red‐winged blackbirds (Figure 3). These results show that while there is a significant species difference in PRLR levels in POA between cowbirds and red‐winged blackbirds, MT treatment can upregulate the expression of PRLR in the POA of brown‐headed cowbirds. However, the MT‐induced increase in PRLR levels in POA was not enough to promote the performance of parental behaviors in cowbirds. In addition, without MT treatment, PRLR abundance in the cowbird POA was substantially lower than both control and MT‐treated red‐winged blackbirds. Thus, we identified a role for hormones activating transcript differences in the brains of these two species and the POA as a target for these activational differences. Altogether, our results suggest that differences in specific brain regions may arise as a consequence of hormonal fluctuations, indicating that the neural mechanisms underlying brood parasitism may involve, at least in part, the activational effects of hormones over the course of development.

With respect to species differences in transcripts abundance (i.e., differences unrelated to MT treatment), we noted that for nearly all transcripts, abundance was higher in the three brain regions in red‐winged blackbirds compared with brown‐headed cowbirds except for MT and PRLR (Figure 3). There was significantly higher MT transcript abundance in the PVN of brown‐headed cowbirds compared with that of red‐winged blackbirds. In contrast, a comparative study of MT immunoreactivity (MT‐ir) in these two species showed more MT‐ir neurons in PVN in red‐winged blackbirds than in cowbirds. 77 The discrepancy between mRNA and protein may be related to post‐transcriptional regulation or differences in the sampling time. For instance, MT‐ir in the PVN remained high in Thai chicken hens incubating eggs, but after nests were removed, MT‐ir abundance decreased significantly. 78 This mechanism may have decreased MT mRNA in PVN in female red‐winged blackbirds that were separated from their nest before testing in our study whereas no such change occurs in brown‐headed cowbirds that are not attached to a nest in the same way. Alternatively, these results may also be related to the regulatory role of MT in egg laying and in enhancing the sensitivity of the oviduct to other peptides needed for regulating reproductive behavior. 79 , 80 This is relevant because cowbirds lay a large number of eggs compared with other birds. 81 While most birds may lay between 3 and 10 eggs in a season and provide care for these eggs, cowbirds continue egg‐laying throughout the breeding season as they are not constrained by parental duties. Thus, elevated levels of MT that facilitate oviposition during the breeding season may be key to the reproductive success of cowbirds. Regardless of the unexpected direction of this difference in MT between brown‐headed cowbirds and red‐winged blackbirds, this differential transcript abundance indicates that the PVN is likely part of the neural pathway that has diverged in the brood parasite.

The other two transcripts investigated here, AVT and GAL, exhibited species differences in the three brain regions. In this case, red‐winged blackbirds had higher transcript abundance compared with brown‐headed cowbirds. With respect to AVT, we observed both overall differences between species as well as differences in the patterns of AVT expression across brain regions. AVT levels in POA were higher than in BST and PVN in red‐winged blackbirds but this pattern is not present in brown‐headed cowbird brains, in which PVN exhibited the highest levels of AVT transcript. We also found that red‐winged blackbird females exhibit higher GAL transcript abundance than brown‐headed cowbirds in the three brain regions; however, the pattern of expression across regions is the same in both species (Figure 3). GAL neurons in POA receive large inputs from vasopressin neurons in PVN 82 ; thus, higher AVT and GAL transcript abundance in red‐winged blackbirds may further enhance the behavioral differences with brood parasites.

Investigating the differences in the co‐expression of these transcripts allowed us to identify sets of transcripts that have diverged in brood parasites as well as to propose a neural pathway where this process has occurred. Analyzing these transcripts as a set may provide greater insight into concordant transcript profiles that share a biological function. We identified many gene sets with positive correlations in POA and especially in PVN of brown‐headed cowbirds. On the other hand, while red‐winged blackbirds exhibited a comparable number of correlated sets in BST to that of brown‐headed cowbirds, AVT and PRLR were not significantly co‐expressed in red‐winged blackbirds as it was in cowbirds. Red‐winged blackbirds also showed fewer co‐expression sets in the POA and PVN compared with cowbirds (Figure 4). Together, the single transcript analysis and the characterization of transcript co‐expression in these three brain regions indicate that all regions, especially the hypothalamic regions, namely POA and PVN, may be part of a neural pathway that has diverged in the cowbird brain, and therefore, require further attention in future studies.

A major caveat of our study involves the difference in collection sites and the time of the breeding season for each species. In the present study, differences in the environment of our species collection sites or the timing in the breeding season in which subjects were collected may result in the observed differences in behavior and transcript abundance reported here. This condition would imply that the differences described in our study were environmental rather than species dependent. While additional studies are needed to discern species‐ and environment‐dependent differences in behavior and transcripts in these two species, it is the case that the results reported here are consistent with the results of previous comparative studies of cowbirds and red‐winged blackbirds from our lab. For instance, we previously reported that cowbirds collected from a population in Texas also avoid nestling‐related stimuli. 36 Likewise, our present finding that the saline‐treated female cowbirds exhibited significantly lower PRLR levels in POA compared with red‐winged blackbirds is consistent with previous results. 35 , 36 , 83 Regarding the time of the breeding season, female red‐winged blackbirds in our study were building nests or incubating eggs at the time of collection, which may have influenced their preference for a nest with eggs as it is possible that if collected later in the breeding season, the preference of female red‐winged blackbirds likely could shift toward nestling‐related stimuli. However, these predictions need to be confirmed. Our interpretation of these results is that MT treatment may enhance already existing preferences in each species. Therefore, MT‐treated red‐winged blackbirds exhibited a preference for eggs, as this would be consistent with the time of the breeding season at the time of collection. However, cowbirds do not go through this stage in the breeding season, and so there is no comparable point in which to compare them to the parental blackbirds that are nest building and incubating. Therefore, their preference for eggs is likely not a result of timing during the breeding season. Moreover, with our current experimental design, we cannot parse out the preference for eggs from the preference for the acoustic stimulus, in this case, the dove coos. In this study, dove coos were used as a positive control sound that has no relevance for either species in the study. While cowbirds parasitize heterospecific nests, dove nests are avoided because doves are unsuitable hosts for cowbirds. 42 , 43 Nonetheless, it is possible that given the lack of availability of suitable hosts, cowbird females are willing to explore and potentially parasitize the nest of less suitable hosts. This hypothesis, however, does not explain the preference observed in red‐winged blackbirds, which are parental species that do not frequent or parasitize the nests of other species.

Given that some form of parental care is typically present in 99% of bird species, 84 it is intriguing that some birds do not provide any further investment in their own offspring after laying an egg. Understanding the evolution of the avian brood parasitic strategy, and the corresponding neural mechanisms underlying this behavior is a long‐standing neuroethological question. Because avian brood parasitism is an evolutionarily derived behavior, the brains of these birds were likely subjected to selective forces that targeted specific neural substrates involved in parental care like the regions that we identified here. It remains to be seen whether other regions in the social behavior neural network and their connectivity have also diverged in these birds. Future studies should also aim to experimentally manipulate these regions and peptides to parse out their contributions to the behavioral phenotypes observed in these birds. Altogether, comparative studies involving avian brood parasites constitute a powerful tool to better understand how changes in neural substrates underlying parental care result in variation in the quality and quantity of these behaviors within and between species.

FUNDING INFORMATION

This project was funded by a National Science Foundation Award #1949188 to KSL.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting Information.

Supplementary Video 1. Interactions with nest contents.

ACKNOWLEDGMENTS

We thank Balcones Wildlife Refuge (Marble Falls, TX) and the USDA at John F. Kennedy Airport (New York, NY) for their help with bird collection. We also thank undergraduate students at Hofstra University for their help with laboratory procedures, including Rosin Crosby, Christian Farrelly, Nina Glawe, Audrey Soo, and Brian Suarez. In addition, we thank Dr, Carlos A. Rodriguez‐Saltos for his work on field experiments and insights into data analysis, as well as Kim Hoke for discussions on data analysis.

DATA AVAILABILITY STATEMENT

Datasets and code are currently available in the Open Science Framework (OSF) platform using this private link (https://osf.io/tgpsw/?view_only=478d047453234e959cf67f4779a9f0d4). Materials will be publicly available once the article is accepted for publication.
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