SARS-CoV-2 surveillance in Norway rats (Rattus norvegicus) from Antwerp sewer system, Belgium.

SARS-CoV-2 human-to-animal transmission can lead to the establishment of novel reservoirs and the evolution of new variants with the potential to start new outbreaks in humans. We tested Norway rats inhabiting the sewer system of Antwerp, Belgium, for the presence of SARS-CoV-2 following a local COVID-19 epidemic peak. In addition, we discuss the use and interpretation of SARS-CoV-2 serological tests on non-human samples. Between November and December 2020, Norway rat oral swabs, faeces and tissues from the sewer system of Antwerp were collected to be tested by RT-qPCR for the presence of SARS-CoV-2. Serum samples were screened for the presence of anti-SARS-CoV-2 IgG antibodies using a Luminex microsphere immunoassay (MIA). Samples considered positive were then checked for neutralizing antibodies using a conventional viral neutralization test (cVNT). The serum of 35 rats was tested by MIA showing three potentially positive sera that were later negative by cVNT. All tissue samples of 39 rats analysed tested negative for SARS-CoV-2 RNA. This is the first study that evaluates SARS-CoV-2 infection in urban rats. We can conclude that the sample of rats analysed had never been infected with SARS-CoV-2. However, monitoring activities should continue due to the emergence of new variants prone to infect Muridae rodents.

Turn taking is not restricted by task specialisation but does not facilitate equality in offspring provisioning.

Sexual conflict arises when two individuals invest in their common offspring because both individuals benefit when their partner invests more. Conditional cooperation is a theoretical concept that could resolve this conflict. Here, parents are thought to motivate each other to contribute to provisioning visits by following the rules of turn taking, which results in equal and efficient investment. However, parents have other tasks besides provisioning, which might hinder taking turns. To investigate restrictions by other care tasks and whether turn taking can be used to match investment, we manipulated brooding duration in female blue tits (Cyanistes caeruleus) during the early nestling phase by changing nest box temperature. As expected, females subjected to cold conditions brooded longer than females under warm conditions. Yet, contrary to our prediction, females had similar visit rates in both treatments, which suggests that females in the cold treatment invested more overall. In addition, the females' turn taking level was higher in the more demanding cold condition (and the calculated randomised turn taking levels of females did not differ), hence females don't seem to be restricted in their turn taking strategy by other care tasks. However, males did not seem to match the females' turn taking levels because they did not adjust their visit rates. Thus, level of turn taking was not restricted by an other sex-specific task in females and did not facilitate a greater investment by their male partners.

A fixed agreement-consequences of brood size manipulation on alternation in blue tits.

Recent studies have proposed that conditional cooperation may resolve sexual conflict over the amount of care provided by each parent. Such conditional cooperation may allow parents to equalize their investment by alternating their provisioning visits. This alternated pattern of male and female visits, that is, alternation, is thought to stimulate each other's investment leading to higher levels of provisioning and potential benefits for offspring development. However, experimental studies testing the role of alternation as an adaptive parental strategy to negotiate the level of investment are still absent. Therefore, we manipulated blue tit (Cyanistes caeruleus) parents by temporarily changing their brood sizes to induce changes in demand and thus visit rates. Parents were expected to visit more-assuming that prey sizes were constant-and alternate at higher levels when confronted with an enlarged brood given the greater potential for sexual conflict. In contrast, in reduced broods visit rates and alternation may become lower due to the smaller investment that is needed for reduced broods. We show that the level of alternation did not differ in response to the manipulated brood sizes, despite a directional change in visit rates for enlarged and reduced broods as expected. Nestlings did not benefit from high levels of alternation as no effects on nestling mass gain were present in either of the different manipulations. These findings indicate that alternation does not serve as a mechanism to motivate each other to feed at higher rates. Parents hence appeared to be inflexible in their level of alternation. We therefore suggest that the level of alternation might reflect a fixed agreement about the relative investment by each of the caring parents.

Enduring rules of care within pairs - how blue tit parents resume provisioning behaviour after experimental disturbance.

Sexual conflict over parental investment can result in suboptimal reproductive output. A recent hypothesis suggests that equality in investment, and hence conflict resolution, may be reached via coordination of parental activities like alternating nest visits. However, how robust patterns of care within couples are against temporal disturbances that create asymmetries in parental investment remains as yet to be shown. We here experimentally created such a social disturbance in a wild population of biparental blue tits (Cyanistes caeruleus) when provisioning their nestlings. We randomly caught and subsequently released one of the parents when nestlings were 6 and 12 days old respectively. On average, the parent that was caught did not resume care for nearly two hours. We then compared the levels of individual investment and within-pair coordination before, during and after the absence of the disturbed parent. We show that the remaining parent partially compensated by increasing its provisioning rate, but this compensatory response was strongest in females when nestlings were 6 days old. Once the caught parent returned to feed its nestlings, both parents resumed provisioning at a similar rate as before the disturbance. Likewise, the within-pair alternation level quickly resembled the pre-manipulated level, independent of nestling age or which sex was caught. Thus our experiment highlights the resilience of parental behaviour against temporal disturbances of individual parents.

How to quantify animal activity from radio-frequency identification (RFID) recordings.

Automated animal monitoring via radio-frequency identification (RFID) technology allows efficient and extensive data sampling of individual activity levels and is therefore commonly used for ecological research. However, processing RFID data is still a largely unresolved problem, which potentially leads to inaccurate estimates for behavioral activity. One of the major challenges during data processing is to isolate independent behavioral actions from a set of superfluous, nonindependent detections. As a case study, individual blue tits (Cyanistes caeruleus) were simultaneously monitored during reproduction with both video recordings and RFID technology. We demonstrated how RFID data can be processed based on the time spent in- and outside a nest box. We then validated the number and timing of nest visits obtained from the processed RFID dataset by calibration against video recordings. The video observations revealed a limited overlap between the time spent in- and outside the nest box, with the least overlap at 23 s for both sexes. We then isolated exact arrival times from redundant RFID registrations by erasing all successive registrations within 23 s after the preceding registration. After aligning the processed RFID data with the corresponding video recordings, we observed a high accuracy in three behavioral estimates of parental care (individual nest visit rates, within-pair alternation and synchronization of nest visits). We provide a clear guideline for future studies that aim to implement RFID technology in their research. We argue that our suggested RFID data processing procedure improves the precision of behavioral estimates, despite some inevitable drawbacks inherent to the technology. Our method is useful, not only for other cavity breeding birds, but for a wide range of (in)vertebrate species that are large enough to be fitted with a tag and that regularly pass near or through a fixed antenna.

Size matters but hunger prevails-begging and provisioning rules in blue tit families.

It is commonly observed in many bird species that dependent offspring vigorously solicit for food transfers provided by their parents. However, the likelihood of receiving food does not only depend on the parental response, but also on the degree of sibling competition, at least in species where parents raise several offspring simultaneously. To date, little is known about whether and how individual offspring adjusts its begging strategy according to the entwined effects of need, state and competitive ability of itself and its siblings. We here manipulated the hunger levels of either the two heaviest or the two lightest blue tit (Cyanistes caeruleus) nestlings in a short-term food deprivation experiment. Our results showed that the lightest nestlings consistently begged more than the heaviest nestlings, an effect that was overruled by the tremendous increase in begging behaviour after food deprivation. Meanwhile, the amplified begging signals after food deprivation were the only cue for providing parents in their decision process. Furthermore, we observed flexible but state-independent begging behaviour in response to changes in sibling need. As opposed to our expectations, nestlings consistently increased their begging behaviour when confronted with food deprived siblings. Overall, our study highlights that individual begging primarily aims at increasing direct benefits, but nevertheless reflects the complexity of a young birds' family life, in addition to aspects of intrinsic need and state.

Decreasing parental task specialization promotes conditional cooperation.

How much to invest in parental care and by who remain puzzling questions fomented by a sexual conflict between parents. Negotiation that facilitates coordinated parental behaviour may be key to ease this costly conflict. However, understanding cooperation requires that the temporal and sex-specific variation in parental care, as well as its multivariate nature is considered. Using a biparental bird species and repeated sampling of behavioural activities throughout a major part of reproduction, we show a clear division of tasks between males and females in provisioning, brooding and foraging. Such behavioural specializations fade with increasing nestling age, which stimulates the degree of alternated feeding visits, as a recently promoted form of conditional cooperation. However, such cooperation is thought to benefit offspring development, which is not supported by our data. Thus, from a proximate point of view, conditional cooperation via alternation critically depends on the division of parental tasks, while the ultimate benefits have yet to be shown.

Structural (UV) and carotenoid-based plumage coloration - signals for parental investment?

Parental care increases parental fitness through improved offspring condition and survival but comes at a cost for the caretaker(s). To increase life-time fitness, caring parents are, therefore, expected to adjust their reproductive investment to current environmental conditions and parental capacities. The latter is thought to be signaled via ornamental traits of the bearer. We here investigated whether pre- and/or posthatching investment of blue tit (Cyanistes caeruleus) parents was related to ornamental plumage traits (UV crown coloration and carotenoid-based plumage coloration) expressed by either the individual itself (i.e. "good parent hypothesis") or its partner (i.e. "differential allocation hypothesis"). Our results show that neither prehatching (that is clutch size and offspring begging intensity) nor posthatching parental investment (provisioning rate, offspring body condition at fledging) was related to an individual's UV crown coloration or to that of its partner. Similar observations were made for carotenoid-based plumage coloration, except for a consistent positive relationship between offspring begging intensity and maternal carotenoid-based plumage coloration. This sex-specific pattern likely reflects a maternal effect mediated via maternally derived egg substances, given that the relationship persisted when offspring were cross-fostered. This suggests that females adjust their offspring's phenotype toward own phenotype, which may facilitate in particular mother-offspring co-adaptation. Overall, our results contribute to the current state of evidence that structural or pigment-based plumage coloration of blue tits are inconsistently correlated with central life-history traits.

Sexually antagonistic selection during parental care is not generated by a testosterone-related intralocus sexual conflict-insights from full-sib comparisons.

The evolution of shared male and female traits can be hampered if selection favours sex-specific optima. However, such genomic conflicts can be resolved when independent male and female mechanisms evolve. The existence, extent and consequences of conflict and/or conflict resolution are currently debated. Endocrinological traits like plasma testosterone (T) are suitable test cases, given their important role in mediating correlated traits, plus their opposing sex-specific fitness effects. We compared full-sibling (brother/sister) captive canaries to test for (1) sexually antagonistic selection characterized by contrasting fitness patterns within pairs of relatives, (2) intersexual genetic correlation of plasma T (h(²) = 0.41 ± 0.31) and (3) intralocus sexual conflict over T levels featured by distinct sex-specific fitness optima. We found potential for sexually antagonistic selection, since high fledgling mass was reached by either brothers or sisters, but not by both. We report a positive intersexual correlation for T, as a requirement for intralocus sexual conflict. However, high levels of T were associated with increased female and decreased male fitness (fledgling mass), which contrasts our expectations and challenges the hypothesis of intralocus sexual conflict driven by T. We hypothesize that behavioural and physiological trade-offs differ between sexes when raising offspring, driving T levels towards a state of monomorphism.

Frequency-dependent selection on female morphs driven by premating interactions with males.

Species showing color polymorphisms-the presence of two or more genetically determined color morphs within a single population-are excellent systems for studying the selective forces driving the maintenance of genetic diversity. Despite a shortage of empirical evidence, it is often suggested that negative frequency-dependent mate preference by males (or diet choice by predators) results in fitness benefits for the rare female morph (or prey type). Moreover, most studies have focused on the male (or predator) behavior in these systems and largely overlooked the importance of female (or prey) resistance behavior. Here, we provide the first explicit test of the role of frequency-dependent and frequency-independent intersexual interactions in female polymorphic damselflies. We identify the stage of the mating sequence when frequency-dependent selection is likely to act by comparing indexes of male mate preference when the female has little (females presented on sticks), moderate (females in cages), and high (females free to fly in the field) ability to avoid male mating attempts. Frequency-dependent male preferences were found only in those experiments where females had little ability to resist male harassment, indicating that premating interactions most likely drive negative frequency-dependent selection in this system. In addition, by separating frequency-dependent male mating preference from the baseline frequency-independent component, we reconcile the seemingly contradictory results of previous studies and highlight the roles of both forms of selection in maintaining the polymorphism at a given equilibrium. We conclude that considering interactions among all players-here, males and females-is crucial to fully understanding the mechanisms underlying the maintenance of genetic polymorphisms in the wild.

Influence of mate preference and laying order on maternal allocation in a monogamous parrot species with extreme hatching asynchrony.

It is well established that in many avian species, prenatal maternal resource allocation varies both between and within clutches and may affect offspring fitness. Differential allocation of maternal resources, in terms of egg weight and yolk composition, may therefore allow the female to adjust brood reduction and to fine-tune reproductive investment in accordance with the expected fitness returns. The adaptive value of such maternal resource allocation is thought to be context-dependent as well as species-specific. We investigated the effects of female preference for her mate on the allocation of prenatal maternal resources in the budgerigar, Melopsittacus undulatus, a monogamous species of parrot that shows an extreme hatching asynchrony. We assessed mate preferences in a two-way preference test and allowed females two breeding rounds: one with the preferred and one with the non-preferred partner. We found no effect of preference on either latency to lay or clutch size, but females mated with the preferred partner laid eggs that contained significantly more yolk. Their eggs also contained significantly more androstenedione but not testosterone. Our results suggest that in this species, female preference may influence maternal resource allocation, and that the functional roles of each androgen in the yolk should be considered separately. In addition, we found a significant effect of laying order on egg and yolk weight as well as on yolk testosterone and androstenedione levels. These measures, however, did not change linearly with the laying order and render it unlikely that female budgerigars compensate for the extreme hatching asynchrony by adjusting within-clutch allocation of prenatal maternal resources.

Differential water mite parasitism, phenoloxidase activity, and resistance to mites are unrelated across pairs of related damselfly species.

Related host species often demonstrate differences in prevalence and/or intensity of infection by particular parasite species, as well as different levels of resistance to those parasites. The mechanisms underlying this interspecific variation in parasitism and resistance expression are not well understood. Surprisingly, few researchers have assessed relations between actual levels of parasitism and resistance to parasites seen in nature across multiple host species. The main goal of this study was to determine whether interspecific variation in resistance against ectoparasitic larval water mites either was predictive of interspecific variation in parasitism for ten closely related species of damselflies (grouped into five "species pairs"), or was predicted by interspecific variation in a commonly used measure of innate immunity (total Phenoloxidase or potential PO activity). Two of five species pairs had interspecific differences in proportions of individuals resisting larval Arrenurus water mites, only one of five species pairs had species differences in prevalence of larval Arrenurus water mites, and another two of five species pairs showed species differences in mean PO activity. Within the two species pairs where species differed in proportion of individuals resisting mites the species with the higher proportion did not have correspondingly higher PO activity levels. Furthermore, the proportion of individuals resisting mites mirrored prevalence of parasitism in only one species pair. There was no interspecific variation in median intensity of mite infestation within any species pair. We conclude that a species' relative ability to resist particular parasites does not explain interspecific variation in parasitism within species pairs and that neither resistance nor parasitism is reflected by interspecific variation in total PO or potential PO activity.

Negative frequency-dependent selection or alternative reproductive tactics: maintenance of female polymorphism in natural populations.

BACKGROUND: Sex-limited polymorphisms have long intrigued evolutionary biologists and have been the subject of long-standing debates. The coexistence of multiple male and/or female morphs is widely believed to be maintained through negative frequency-dependent selection imposed by social interactions. However, remarkably few empirical studies have evaluated how social interactions, morph frequencies and fitness parameters relate to one another under natural conditions. Here, we test two hypotheses proposed to explain the maintenance of a female polymorphism in a species with extreme geographical variation in morph frequencies. We first elucidate how fecundity traits of the morphs vary in relation to the frequencies and densities of males and female morphs in multiple sites over multiple years. Second, we evaluate whether the two female morphs differ in resource allocation among fecundity traits, indicating alternative tactics to maximize reproductive output. RESULTS: We present some of the first empirical evidence collected under natural conditions that egg number and clutch mass was higher in the rarer female morph. This morph-specific fecundity advantage gradually switched with the population morph frequency. Our results further indicate that all investigated fecundity traits are negatively affected by relative male density (i.e. operational sex ratio), which confirms male harassment as selective agent. Finally, we show a clear trade-off between qualitative (egg mass) and quantitative (egg number) fecundity traits. This trade-off, however, is not morph-specific. CONCLUSION: Our reported frequency- and density-dependent fecundity patterns are consistent with the hypothesis that the polymorphism is driven by a conflict between sexes over optimal mating rate, with costly male sexual harassment driving negative frequency-dependent selection on morph fecundity.

Biogeographical survey identifies consistent alternative physiological optima and a minor role for environmental drivers in maintaining a polymorphism.

The contribution of adaptive mechanisms in maintaining genetic polymorphisms is still debated in many systems. To understand the contribution of selective factors in maintaining polymorphism, we investigated large-scale (>1000 km) geographic variation in morph frequencies and fitness-related physiological traits in the damselfly Nehalennia irene. As fitness-related physiological traits, we investigated investment in immune function (phenoloxidase activity), energy storage and fecundity (abdomen protein and lipid content), and flight muscles (thorax protein content). In the first part of the study, our aim was to identify selective agents maintaining the large-scale spatial variation in morph frequencies. Morph frequencies varied considerably among populations, but, in contrast to expectation, in a geographically unstructured way. Furthermore, frequencies co-varied only weakly with the numerous investigated ecological parameters. This suggests that spatial frequency patterns are driven by stochastic processes, or alternatively, are consequence of highly variable and currently unidentified ecological conditions. In line with this, the investigated ecological parameters did not affect the fitness-related physiological traits differently in both morphs. In the second part of the study, we aimed at identifying trade-offs between fitness-related physiological traits that may contribute to the local maintenance of both colour morphs by defining alternative phenotypic optima, and test the spatial consistency of such trade-off patterns. The female morph with higher levels of phenoloxidase activity had a lower thorax protein content, and vice versa, suggesting a trade-off between investments in immune function and in flight muscles. This physiological trade-off was consistent across the geographical scale studied and supports widespread correlational selection, possibly driven by male harassment, favouring alternative trait combinations in both female morphs.

Frequency-dependent variation in mimetic fidelity in an intraspecific mimicry system.

Contemporary theory predicts that the degree of mimetic similarity of mimics towards their model should increase as the mimic/model ratio increases. Thus, when the mimic/model ratio is high, then the mimic has to resemble the model very closely to still gain protection from the signal receiver. To date, empirical evidence of this effect is limited to a single example where mimicry occurs between species. Here, for the first time, we test whether mimetic fidelity varies with mimic/model ratios in an intraspecific mimicry system, in which signal receivers are the same species as the mimics and models. To this end, we studied a polymorphic damselfly with a single male phenotype and two female morphs, in which one morph resembles the male phenotype while the other does not. Phenotypic similarity of males to both female morphs was quantified using morphometric data for multiple populations with varying mimic/model ratios repeated over a 3 year period. Our results demonstrate that male-like females were overall closer in size to males than the other female morph. Furthermore, the extent of morphological similarity between male-like females and males, measured as Mahalanobis distances, was frequency-dependent in the direction predicted. Hence, this study provides direct quantitative support for the prediction that the mimetic similarity of mimics to their models increases as the mimic/model ratio increases. We suggest that the phenomenon may be widespread in a range of mimicry systems.

Thermal plasticity in life-history traits in the polymorphic blue-tailed damselfly, Ischnura elegans: no differences between female morphs.

Female polymorphism is observed in various animal species, but is particularly common in damselflies. The maintenance of this polymorphism has traditionally been explained from frequency and density dependent sexual conflict, however, the role of abiotic factors has recently attracted more interest. Here, the role of ambient temperature in shaping life-history was investigated for the three female morphs of Ischnura elegans (Vander Linden) (Zygoptera: Coenagrionidae). Eggs were obtained from the three mature female morphs for two populations in the Netherlands. Using a split-brood design, eggs of both populations were divided between a cold and a warm treatment group in the laboratory, and egg survival and hatching time were measured. Significant thermal plasticity was found in both hatching time and egg survival between both temperature treatments. However, individuals born to mothers belonging to different colour morphs did not differ in their response to temperature treatment. Independent of colour morph, clear differences in both life-history traits between the populations were found, suggesting local adaptation. Specifically, individuals from one population hatched faster but had lower egg survival in both thermal regimes. The selection force establishing fast hatching could be (facultative) bivoltinism in one of the populations compared to univoltinism in the other. This would be in line with the more southern (and more coastal) location of the presumed bivoltine population and the inverse relation between voltinism and latitude known from earlier studies. However, other natural selection forces, e.g. deterioration of the aquatic habitat, may also drive fast hatching.