Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Abstracts

ABSTRACT

The normal development of empathy has been proposed to be imperative to the healthy emotional and social functioning of youths. In contrast, compromised levels of empathy have been linked to an increased propensity to engage in antisocial behaviors, including animal cruelty. Previous findings have revealed parent attachment to be intrinsically linked to the development of empathy. This association has been shown to play a role in predicting the expression of various outcome behaviors, including both those which are prosocial in nature, and those which are antisocial, and potentially aggressive. This study examines these associations in a sample of 281 12- to 18-year-old students. The aims included the investigation of the direct predictive roles played by attachment and empathy for prosocial and antisocial behaviors directed at both humans and animals. We also investigated the mediating role played by empathy in these relationships. Attachment and empathy significantly predicted prosocial and antisocial behaviors, both individually, and in combination. Furthermore, empathy was found to serve a mediating role in the associations between attachment and: human-directed prosocial behavior, the humane treatment of animals, and animal cruelty. These findings expand upon existing literature by demonstrating that it is, at least partially, through empathy that attachment to parents predicts prosocial and antisocial behaviors during adolescence. This is in contrast to the direction of relationships implied by some previous findings and proposals, which have suggested that treating animals humanely fosters the normal development of empathy, for example. Notwithstanding the promising findings revealed by the current study, we recommend that replicating this research using a larger sample will assist in addressing the limited generalizability identified in the current study. It is further proposed that the use of a lie scale could limit the influence of social desirability responding. Future research is also needed to determine the direction of the demonstrated relationships.     

Linking behavioral states to landscape features for improved conservation management

1. A central theme for conservation is understanding how animals differentially use, and are affected by change in, the landscapes they inhabit. However, it has been challenging to develop conservation schemes for habitat‐specific behaviors.
2. Here we use behavioral change point analysis to identify behavioral states of golden eagles (Aquila chrysaetos) in the Sonoran and Mojave Deserts of the southwestern United States, and we identify, for each behavioral state, conservation‐relevant habitat associations.
3. We modeled behavior using 186,859 GPS points from 48 eagles and identified 2,851 distinct segments comprising four behavioral states. Altitude above ground level (AGL) best differentiated behavioral states, with two clusters of short‐distance movement behaviors characterized by low AGL (state 1 AGL = 14 m (median); state 2 AGL = 11 m) and two associated with longer‐distance movement behaviors and characterized by higher AGL (state 3 AGL = 108 m; state 4 AGL = 450 m).
4. Behaviors such as perching and low‐altitude hunting were associated with short‐distance movements in updraft‐poor environments, at higher elevations, and over steeper and more north‐facing terrain. In contrast, medium‐distance movements such as hunting and transiting were over gentle and south‐facing slopes. Long‐distance transiting occurred over the desert habitats that generate the best updraft.
5. This information can guide management of this species, and our approach provides a template for behavior‐specific habitat associations for other species of management concern.

     

Mantis Shrimp Navigate Home Using Celestial and Idiothetic Path Integration

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Nonlinear analysis of heart rate variability for evaluating the growing pig stress response to an acute heat episode

Heart rate variability (HRV) is a proxy measure of autonomic function and can be used as an indicator of swine stress. While traditional linear measures are used to distinguish between stressed and unstressed treatments, inclusion of nonlinear HRV measures that evaluate data structure and organization shows promise for improving HRV interpretation. The objective of this study was to evaluate the inclusion of nonlinear HRV measures in response to an acute heat episode. Twenty 12- to 14-week-old growing pigs were individually housed for 7 days and acclimated to thermoneutral conditions (20.35°C ± 0.01°C; 67.6% ± 0.2% RH) before undergoing one of the two treatments: (1) thermoneutral control (TN; n = 10 pigs) or (2) acute heat stress (HS; n = 10 pigs; 32.6°C ± 0.1°C; 26.2% ± 0.1% RH). In Phase 1 of the experimental procedure (P1; 60 min), pigs underwent a baseline HRV measurement period in thermoneutral conditions before treatment [Phase 2; P2; 60 min once gastrointestinal temperature (T_g) reached 40.6°C], where HS pigs were exposed to heated conditions and TN pigs remained in thermoneutral conditions. After P2, all pigs were moved back to thermoneutral conditions (Phase 3; P3; 60 min). During each phase, T_g data were collected every 5 min and behavioural data were collected to evaluate the amount of time each pig spent in an active posture. Additionally, linear (time and frequency domain) and nonlinear [sample entropy (SampEn), de-trended fluctuation analysis, percentage recurrence, percentage determinism (%DET), mean diagonal line length in a recurrence plot] HRV measures were quantified. Heat stressed pigs exhibited greater T_g (P = 0.002) and spent less time in an active posture compared to TN pigs during P2 (P = 0.0003). Additionally, low frequency to high frequency ratio was greater in HS pigs during P3 compared to TN pigs (P = 0.02). SampEn was reduced in HS pigs during P2 (P = 0.01) and P3 (P = 0.03) compared to TN pigs. Heat stressed pigs exhibited greater %DET during P3 (P = 0.03) and tended to have greater %DET (P = 0.09) during P2 than TN pigs. No differences between treatments were detected for the remaining HRV measures. In conclusion, linear HRV measures were largely unchanged during P2. However, changes to SampEn and %DET suggest increased heat stress as a result of the acute heat episode. Future work should continue to evaluate the benefits of including nonlinear HRV measures in HRV analysis of swine heat stress.     

The role of cross-border transhumance in influencing resident herders’ cattle husbandry practices and use of genetic resources

For centuries, the humid West African coastal country of Benin attracts cattle herders from neighboring Sahelian countries such as Niger, Burkina Faso and Nigeria. Each year, several thousands of cattle are trekked over hundreds of kilometers to Benin following established and non-established transhumance corridors. This cross-border mobility has been shown to play an important role in ensuring the productivity of the transhumant herds through adequate late dry season access to pastoral resources. Yet, its effects on the traditional agro-pastoral production systems that are encountered along the routes in Benin have never been explicitly investigated. Therefore, we collected socio-economic household data, herd characteristics and management data from 104 resident herders and 38 transhumant herders in 2 vegetation zones of Benin. To determine whether or not the proximity to transhumance corridors affects sedentary production systems, characteristics of cattle herds and farmers’ management practices were compared between villages close to (within a 25 km buffer) and far from (outside a 25 km buffer) transhumance corridors within and between vegetation zones using non-parametric statistical tests. Existing relationships between resident and transhumant herders were also identified and characterized. Subsequently, herd characteristics and management practices were compared between resident herders having relationships with transhumant herders and those without. Herd sizes of resident herders living close to transhumance corridors were larger (P < 0.01) than those of their counterparts living far away. Also, proximity to transhumance corridors had positive effects on herd management practices. The relationships between resident and transhumant herders were governed by a variety of interests including encampment/manuring contracts, exploitation of grazing lands and watering points, trading and bartering of cattle. This exchange of cattle is an important driver of change in the breed composition of local herds and represents an opportunity for resident herders to enhance their herds’ productivity through crossbreeding. However, the mere replacement or indiscriminate crossbreeding of local cattle breeds with those kept by transhumant herders threatens the sustainability of the traditional resident herding systems by increasing the risk of genetic erosion and loss of valuable adaptive traits in indigenous animal genetic resources.     

Endozoochory of a dry‐fruited tree aids quarry passive restoration and seed soaking further increases seedling emergence

As practitioners promote passive restoration as a complementary approach to technical reclamation, it is imperative to know its drivers. Although the consequences of endozoochory can be crucial to passive restoration success, few experimental studies assess the use of heavily disturbed sites by seed dispersers such as carnivores and how the seeds they bring in emerge and survive. Using an indoor sowing experiment conducted in a quarry located within a natural park in Portugal, we examined for the first time how carnivore endozoochorous seeds collected in the quarry potentially influence its passive restoration, through effects on plant emergence and survival. Also, we tested whether sowing date and water soaking, relevant factors when sowings are to be carried out, would affect seedling emergence and mortality rates when compared with the effect of endozoochory. Our target species were included in the revegetation plan of the quarry, of which endozoochorous seeds of Carob tree (Ceratonia siliqua) were collected in sufficient number for analysis. Irrespective of the carnivore species, endozoochorous carob seeds performed similarly to untreated seeds regarding emergence rates. Endozoochorous carob seedlings showed greater mortality rates but the net result for the plant can still be the colonization of recently vacant habitats by a large proportion of viable seeds. Concerning sowing date, the later carob seeds were sown over the fruit‐ripening season the faster seedlings emerged. Water soaking increased emergence rate by 6.5 times. Broadly, sowings with previous soaking and carnivore‐mediated seed dispersal of this dry‐fruited tree can jointly enhance quarry restoration.     

Towards the integration of animal‐borne instruments into global ocean observing systems

Marine animals are increasingly instrumented with environmental sensors that provide large volumes of oceanographic data. Here, we conduct an innovative and comprehensive global analysis to determine the potential contribution of animal‐borne instruments (ABI) into ocean observing systems (OOSs) and provide a foundation to establish future integrated ocean monitoring programmes. We analyse the current gaps of the long‐term Argo observing system (>1.5 million profiles) and assess its spatial overlap with the distribution of marine animals across eight major species groups (tuna and billfishes, sharks and rays, marine turtles, pinnipeds, cetaceans, sirenians, flying seabirds and penguins). We combine distribution ranges of 183 species and satellite tracking observations from >3,000 animals. Our analyses identify potential areas where ABI could complement OOS. Specifically, ABI have the potential to fill gaps in marginal seas, upwelling areas, the upper 10 m of the water column, shelf regions and polewards of 60° latitude. Our approach provides the global baseline required to plan the integration of ABI into global and regional OOS while integrating conservation and ocean monitoring priorities.     

植物大年结实及其与动物贮食行为之间的关系

大年结实(mast seeding)是多生年植物种群周期性同步大量繁殖的一种自然现象。大年结实作为植物适应环境条件、提高繁殖能力的一种策略而备受关注, 但其驱动机制和进化意义尚存在较大争议。在依赖动物扩散种子的植物中, 大年结实被认为是一种调控动物贮食行为、提高种子扩散效率, 并最终增加繁殖成功率的一种策略; 动物介导的植物间互作可能是促进植物共存的进化驱动力。本文简要梳理了大年结实现象的各种假说, 提出了一个包括气候、资源、动植物互作的理解大年结实机制的概念框架, 并着重讨论了大年结实和动物贮食行为之间的关系及其进化和生态意义。建议未来研究需要借助长期生态监测和分子生物学方法, 揭示植物大年结实与动物贮食行为之间的生态与进化过程。