Syndromic Surveillance 2.0: Emerging Global Surveillance Strategies for Infectious Disease Epidemics.

The U.S. Army has a long history of preventing, detecting, and treating infectious diseases. Like other organizations and agencies involved in public health, the Army is increasingly interested in syndromic surveillance strategies-those designed to identify outbreaks before clinical data are available. Researchers use various methods to identify surveillance strategies across the globe, investigate these strategies' benefits and limitations, and recommend actions to aid the Army in their efforts to detect emerging epidemics and pandemics.

AOPERA: A proposed methodology and inventory of effective tools to link chemicals to adverse outcome pathways.

New approaches, like the Adverse Outcome Pathway (AOP) framework, have been developed to describe how chemicals cause toxicity by linking in vitro assays to adverse health outcomes. However, approaches, tools and resources for development of AOPs have not been well described. Here we review information resources for AOP development and define a streamlined process for linking a chemical to an existing AOP. We propose a four step process to facilitate AOP development: link the uncharacterized chemical directly to Molecular Initiating Events, Key Events, or Adverse Outcomes; identify analogs with toxicological information for the uncharacterized chemical; link the characterized chemical (initial chemical if characterized, a characterized analog if initial chemical is not) to Molecular Initiating Events, Key Events, or Adverse Outcomes; and identify AOPs that contain the Molecular Initiating Events, Key Events, or Adverse Outcomes that were found in Steps 1 and 3. The process and library of informational resources proposed and tested here served as the foundation for an informational online tool (AOPERA) that helps practitioners identify their current-state knowledge gaps, navigate the four-step process, and connect to relevant resources. AOPERA can be found at https://igbb.github.io/AOPERA_HTML. Additionally, we anticipate that by simplifying and standardizing the process of linking a chemical to a known AOP, we will lower the barrier to entry for this objective and increase its accessibility to new practitioners. In turn, this may increase the demand for new or improved AOPs to which practitioners can link chemicals, thereby contributing to the expansion of the library of known AOPs.

A modular approach for assembly of quantitative adverse outcome pathways.

The adverse outcome pathway (AOP) framework is a conceptual construct that mechanistically links molecular initiating events to adverse biological outcomes through a series of causal key events (KEs) that represent the perturbation of the biological system. Quantitative, predictive AOPs are necessary for screening emerging contaminants and potential substitutes to inform their prioritization for testing. In practice, they are not widely used because they can be costly to develop and validate. A modular approach for assembly of quantitative AOPs, based on existing knowledge, would allow for rapid development of biological pathway models to screen contaminants for potential hazards and prioritize them for subsequent testing and modeling. For each pair of KEs, a quantitative KE relationship (KER) can be derived as a response-response function or a conditional probability matrix describing the anticipated change in a KE based on the response of the prior KE. This transfer of response across KERs can be used to assemble a quantitative AOP. Here we demonstrate the use of proposed approach in two cases: inhibition of cytochrome P450 aromatase leading to reduced fecundity in fathead minnows and ionic glutamate receptor mediated excitotoxicity leading to memory impairment in rodents. The model created from these chains have value in characterizing the pathway and the potential or relative level of toxicological effect anticipated. This approach to simplistic, modular AOP models has wide applicability for rapid development of biological pathway models.

Quantifying Wildlife and Navigation Benefits of a Dredging Beneficial-Use Project in the Lower Atchafalaya River: A Demonstration of Engineering with Nature®.

The US Army Corps of Engineers (USACE) operates and maintains numerous projects in support of its various civil works missions including flood damage risk reduction, navigation, and ecosystem restoration. Originally authorized on an economic basis, these projects may produce a broad array of unaccounted for ecosystem services (ESs) that contribute to overall human, societal, and environmental well-being. Efforts are underway to capture the full array of environmental, economic, and social impacts of these projects. Methods are needed to identify relevant ESs generated by these nature-based projects and to measure their contribution to societal well-being with an emphasis placed on use of readily available data. Performance metrics were collected to capture the benefits of strategic placement of dredged material in river systems to allow formation of islands that produce a wide array of ESs. These performance metrics can be converted to ESs with market value or combined in a decision analytical approach to demonstrate the relative gain in utility. This approach is demonstrated on a riverine island created on the Atchafalaya River, Louisiana, as a result of the strategic placement of dredged material. The outcomes foster integration of ES assessment into project design and management practices and support more comprehensive project evaluation and widespread application. Integr Environ Assess Manag 2018;14:759-768. Published 2018. This article is a US Government work and is in the public domain in the USA.

An enhanced adaptive management approach for remediation of legacy mercury in the South River.

Uncertainties about future conditions and the effects of chosen actions, as well as increasing resource scarcity, have been driving forces in the utilization of adaptive management strategies. However, many applications of adaptive management have been criticized for a number of shortcomings, including a limited ability to learn from actions and a lack of consideration of stakeholder objectives. To address these criticisms, we supplement existing adaptive management approaches with a decision-analytical approach that first informs the initial selection of management alternatives and then allows for periodic re-evaluation or phased implementation of management alternatives based on monitoring information and incorporation of stakeholder values. We describe the application of this enhanced adaptive management (EAM) framework to compare remedial alternatives for mercury in the South River, based on an understanding of the loading and behavior of mercury in the South River near Waynesboro, VA. The outcomes show that the ranking of remedial alternatives is influenced by uncertainty in the mercury loading model, by the relative importance placed on different criteria, and by cost estimates. The process itself demonstrates that a decision model can link project performance criteria, decision-maker preferences, environmental models, and short- and long-term monitoring information with management choices to help shape a remediation approach that provides useful information for adaptive, incremental implementation.

A weight of evidence approach for hazard screening of engineered nanomaterials.

Hazard identification is an important step in assessing nanomaterial risk and is required under multiple regulatory frameworks in the US, Europe and worldwide. Given the emerging nature of the field and complexity of nanomaterials, multiple studies on even basic material properties often result in varying data pointing in different directions when data interpretation is attempted. Weight of evidence (WOE) evaluation has been recommended for nanomaterial risk assessment, but the majority of WOE frameworks are qualitative in nature and do not satisfy the growing needs for objectivity and transparency that are necessary for regulatory decision making. This paper implements a quantitative WOE framework that utilizes multi-criteria decision analysis methodology for integrating individual studies on nanomaterial hazard resulting from physico-chemical and toxicological properties of nanomaterials. For the first time, a WOE approach explicitly integrates expert evaluation of data quality of available information. Application of the framework is illustrated for titanium dioxide nanoparticles (nano-TiO2), but the approach is designed to compare the relative hazard of several nanomaterials as well as emerging stressors in general.

Enhanced adaptive management: integrating decision analysis, scenario analysis and environmental modeling for the Everglades.

We propose to enhance existing adaptive management efforts with a decision-analytical approach that can guide the initial selection of robust restoration alternative plans and inform the need to adjust these alternatives in the course of action based on continuously acquired monitoring information and changing stakeholder values. We demonstrate an application of enhanced adaptive management for a wetland restoration case study inspired by the Florida Everglades restoration effort. We find that alternatives designed to reconstruct the pre-drainage flow may have a positive ecological impact, but may also have high operational costs and only marginally contribute to meeting other objectives such as reduction of flooding. Enhanced adaptive management allows managers to guide investment in ecosystem modeling and monitoring efforts through scenario and value of information analyses to support optimal restoration strategies in the face of uncertain and changing information.

Environmental engineering of navigation infrastructure: a survey of existing practices, challenges, and potential opportunities.

Navigation infrastructure such as channels, jetties, river training structures, and lock-and-dam facilities are primary components of a safe and efficient water transportation system. Planning for such infrastructure has until recently involved efforts to minimize impacts on the environment through a standardized environmental assessment process. More recently, consistent with environmental sustainability concepts, planners have begun to consider how such projects can also be constructed with environmental enhancements. This study examined the existing institutional conditions within the US Army Corps of Engineers and cooperating federal agencies relative to incorporating environmental enhancements into navigation infrastructure projects. The study sought to (1) investigate institutional attitudes towards the environmental enhancement of navigation infrastructure (EENI) concept, (2) identify potential impediments to implementation and solutions to such impediments, (3) identify existing navigation projects designed with the express intent of enhancing environmental benefit in addition to the primary project purpose, (4) identify innovative ideas for increasing environmental benefits for navigation projects, (5) identify needs for additional technical information or research, and (6) identify laws, regulations, and policies that both support and hinder such design features. The principal investigation tool was an Internet-based survey with 53 questions. The survey captured a wide range of perspectives on the EENI concept including ideas, concerns, research needs, and relevant laws and policies. Study recommendations included further promotion of the concept of EENI to planners and designers, documentation of existing projects, initiation of pilot studies on some of the innovative ideas provided through the survey, and development of national goals and interagency agreements to facilitate implementation.

Variable vitellogenin response of Japanese medaka (Oryzias latipes) to weekly estrogen exposure.

Japanese medaka (Oryzias latipes) is a valuable model organism in reproductive and developmental toxicity testing. The purpose of this experiment is to assess the response of medaka to aquatic estrogen exposure over the course of 1 year. Each week, three pairs of adult male medaka were exposed separately for 4 days (100% static renewal daily) to 17beta-estradiol at a nominal level of 25 microg/l, with a fourth pair of fish exposed separately to an ethanol control. Vitellogenin (VTG) induction was observed each week, with hepatic and plasma VTG levels significantly higher (P < 0.001) than reported for ethanol control specimens. A significant (P < 0.001) increasing trend was observed for plasma VTG results over the duration of the study, whereas a decreasing trend (P = 0.030) of hepatic VTG was evident. A Durbin-Watson test, however, did not demonstrate any serial autocorrelation of hepatic (d = 1.180) or plasma (d = 1.311) VTG levels over the duration of the study. Time-series transformations of the hepatic and plasma VTG data did not reveal any significant seasonal or behavioral patterns. However, significant intermittent peaks in VTG production were observed in both tissue types during the study. These data indicate that some consideration must be taken to time long-term medaka exposures (>20 weeks) in order to eliminate any influence of cyclic changes on plasma VTG response. Alternatively, hepatic cytosolic measurement of VTG appears to show a more sensitive response to aquatic estrogen exposure.

Quantification of whole brain arginine vasotocin for two Syngnathus pipefishes: elevated concentrations correlated with paternal brooding.

Arginine vasotocin (AVT) regulates many aspects of fish physiology and behavior including courtship and reproduction. This study begins to address whether paternal functions for AVT have independently evolved in species in which paternal behavior has evolved. We quantified AVT in the brains of Syngnathus fuscus and Syngnathus floridae which, like other pipefishes and seahorses of the family Syngnathidae, brood embryos within specialized structures of the male. For both species, we measured AVT concentrations seventeenfold higher for some males during brooding compared to post-brooding males. Comparable whole brain AVT concentrations between gravid females and males with broods at some embryonic development stages suggest physiological similarities that we hypothesize is related to nutrient provisioning but should be elucidated with further studies including a detailed anatomical analysis of AVT production. Earlier studies have identified differences in the brooding structures of these species. Here we documented interspecific differences in the variability and mean AVT concentration for non-brooding males, the brood stage showing a return to post-brooding concentrations, and the variability of AVT concentrations for brooding males with embryos in some development stages. Future investigations should use these data to investigate the potential for divergent AVT function between species, sexes, and brooding males with embryos of different developmental stages.

Elevated whole brain arginine vasotocin with Aroclor 1254 exposure in two Syngnathus pipefishes.

The neurohypophysial hormone arginine vasotocin (AVT) and the neuroendocrine system strongly interact with the rest of the teleostean endocrine system. The aim of this study is to investigate the effects of exposure to an endocrine disruptor on whole brain AVT concentrations for the pipefishes Syngnathus floridae and S. fuscus. Following treatment over the entire brood period, AVT concentrations were significantly higher for Aroclor 1254-exposed, post-brooding males compared to controls for both species. Considering both previously documented seventeen-fold increases in AVT for brooding males with embryos in some developmental stages and changes in parental nutrient concentrations after Aroclor 1254 exposure, these data begin to address potential physiological mechanisms that may underlie paternal activities in syngnathid males.

Multiple stressor effects of methoprene, permethrin, and salinity on limb regeneration and molting in the mud fiddler crab (Uca pugnax).

Exposure to multiple stressors from natural and anthropogenic sources poses risk to sensitive crustacean growth and developmental processes. Applications of synthetic pyrethroids and insect growth regulators near shallow coastal waters may result in harmful mixture effects depending on the salinity regime. The potential for nonadditive effects of a permethrin (0.01 2 gg/L), methoprene (0.03-10 tg/L ), and salinity (10-40 ppt) exposure on male and female Uca pugnax limb regeneration and molting processes was evaluated by employing a central composite rotatable design with multifactorial regression. Crabs underwent single-limb autotomy followed by a molting challenge under I of 16 different mixture treatments. During the exposure (21-66 d), individual limb growth, major molt stage duration, abnormal limb regeneration, and respiration were monitored. At 6 d postmolt, changes in body mass, carapace width, and body condition factor were evaluated. Dorsal carapace tissue was collected, and protein and chitin were extracted to determine the composition of newly synthesized exoskeleton. The present results suggest chronic, low-dose exposures to multiple pesticide stressors cause less-than-additive effects on U. pugnax growth processes. Under increasing concentrations of methoprene and permethrin, males had more protein in their exoskeletons and less gain in body mass, carapace width, and body condition compared to females. Females exhibited less gain in carapace width than controls in response to methoprene and permethrin. Females also displayed elevated respiration rates at all stages of molt, suggesting a high metabolic rate. Divergent growth and fitness between the sexes over the long term could influence crustacean population resilience.

Direct evidence for embryonic uptake of paternally-derived nutrients in two pipefishes (Syngnathidae: Syngnathus spp.).

Seahorses, sea dragons and pipefishes of the teleost family Syngnathidae are unique in that embryos develop within specialized brooding structures of the male. We enriched brooding Syngnathus fuscus and Syngnathus floridae males with injections of L-lysine-[(15)N(2)] and 16:0-palmitic acid 1-[(13)C] to demonstrate embryonic uptake of paternally-derived nutrients. While all embryos demonstrated amino acid enrichment, late stages showed significantly higher [(15)N], indicating greater utilization of paternal resources as yolk reserves diminished and embryonic energy demands increased. Limited embryonic [(13)C] uptake, defined as less than 10% of adult enrichment, in 75 and 81% of S. floridae and S. fuscus respectively signified rapid lipid metabolism and thus the need for greater enrichment. Interspecific differences in embryonic uptake of paternally-derived nutrients were not demonstrated. However, interspecific differences in egg nutrient reserves and fry size but comparable fry nutrient levels along with data from a published paternal exposure study indicate paternal transfer in S. fuscus most likely compensates for the comparative egg nutrient deficiency. This study is the first to our knowledge to provide direct evidence for the functional significance of the brood pouch in nutrient provisioning. These results add comparative information on the diversity of Syngnathid paternal care and further our understanding of paternal influence on development.

Utilization of protein expression profiles as indicators of environmental impairment of smallmouth bass (Micropterus dolomieu) from the Shenandoah River, Virginia, USA.

The Shenandoah River (VA, USA), the largest tributary of the Potomac River (MD, USA) and an important source of drinking water, has been the site of extensive fish kills since 2004. Previous investigations indicate environmental stressors may be adversely modulating the immune system of smallmouth bass (Micropterus dolomieu) and other species. Anterior kidney (AK) tissue, the major site of blood cell production in fish, was collected from smallmouth bass at three sites along the Shenandoah River. The tissue was divided for immune function and proteomics analyses. Bactericidal activity and respiratory burst were significantly different between North Fork and mainstem Shenandoah River smallmouth bass, whereas South Fork AK tissue did not significantly differ in either of these measures compared with the other sites. Cytotoxic cell activity was highest among South Fork and lowest among North Fork AK leukocytes. The composite two-dimension gels of the North Fork and mainstem smallmouth bass AK tissues contained 584 and 591 spots, respectively. South Fork smallmouth bass AK expressed only 335 proteins. Nineteen of 50 proteins analyzed by matrix-assisted laser desorption ionization-time of flight were successfully identified. Three of the four identified proteins with increased expression in South Fork AK tissue were involved in metabolism. Seven proteins exclusive to mainstem and North Fork smallmouth bass AK and expressed at comparable abundances serve immune and stress response functions. The proteomics data indicate these fish differ in metabolic capacity of AK tissue and in the ability to produce functional leukocytes. The variable responses of the immune function assays further indicate disruption to the immune system. Our results allow us to hypothesize underlying physiological changes that may relate to fish kills and suggest relevant contaminants known to produce similar physiological disruption.

Limb regeneration and molting processes under chronic methoprene exposure in the mud fiddler crab, Uca pugnax.

Insect growth regulator application for wetland mosquito control remains controversial due to the potential for disruption of normal development and growth processes in non-target crustaceans and beneficial arthropods, e.g. Apis mellifera. Concerns include slow-release methoprene formulations and its environmental breakdown products which mimic an endogenous crustacean hormone and retinoids, respectively. Our primary objective was to evaluate the effect that a chronic methoprene exposure would have on male and female Uca pugnax limb regeneration and molting. After single limb autonomy, limb growth and molt stage were monitored every two days while eyestalk ablation was used to induce proecdysis. Dorsal carapace was collected 6 days post-molt to determine protein and chitin content. In post-molt crabs, methoprene-exposed individuals displayed lower percent gain in body weight. Male crabs lost more weight per body volume than females, took significantly longer to proceed through proecdysis than females exposed to 0.1 microg/L methoprene and exhibited significantly elevated frequency for abnormal limb formation at 1.0 microg/L while females displayed no such trend. Methoprene did not significantly alter extractable exoskeleton protein or chitin content. However, variable water-soluble protein expression increased with exposure at 1.0 microg/L (1 ppb) which contributed to overall variability in total protein content. Our findings suggest that adult male U. pugnax possess greater sensitivity to chronic methoprene exposure during limb regeneration and molting, potentially affecting their post-molt fitness. Furthermore, methoprene has the potential to impact post-molt biomass and exocuticle quality.

No acute toxicity to Uca pugnax, the mud fiddler crab, following a 96-h exposure to sediment-bound permethrin.

In coastal areas, the application of pyrethroid insecticides and the resulting sediment residues pose a potential threat to marine benthic ecosystems. Pyrethroids cause acute toxicity and exhibit a wide range of sublethal effects on fish and crustaceans when exposure is aqueous. Fiddler crabs that inhabit salt marsh sediment are sensitive to sediment-associated pollutants and serve as a sentinel species for xenobiotic exposure. We exposed adult U. pugnax to salt marsh sediment spiked with different 60% trans/40% cis permethrin concentrations for 96 h, and evaluated changes in oxygen consumption rate, hemolymph osmolarity, and glutathione S-transferase activity (GST) following exposure. Marsh sediment was not lethal to U. pugnax at permethrin concentrations of 100-10,000 microg/kg. Sediment-bound permethrin had no significant effect on respiration and osmoregulation. Exposure caused an induction of hepatopancreas GST in a dose-dependent manner. Gill and midgut tissues showed induction at permethrin concentrations at 10,000 microg/kg. We conclude that short term exposure to permethrin-contaminated sediment does not pose a significant threat to this species or impact respiration and osmoregulation. Furthermore, increased GST activity allows us to evaluate this enzyme's induction as a generalist biomarker for sediment-bound pyrethroid exposures.

Influence of persistent contaminants and steroid hormones on glioblastoma cell growth.

Glioblastoma multiforme (GBM), a malignancy characterized by its rapid progression, presents a lower risk of occurrence in women during their reproductive years. Necrosis of brain tissue during tumor invasion releases free lipids, and therefore might release contaminants stored in phospholipid-rich neuronal tissue. This study assesses the growth response of two human glioblastoma cell lines, T98G and U138-MG, treated with environmental chemicals known or likely to persist within the brain. Persistent chlorinated pesticides, industrial contaminants, persistent perfluorinated chemicals, and steroid hormones were assayed over a range of concentrations. Although cytotoxic effects were seen in both T98G and U138-MG cells, proliferative responses occurred only in the T98G cell line. Dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), and polychlorinated biphenyl (PCB) 153 were cytotoxic in both lines at 5000 nM. Perfluorodecanoic acid (PFDA), perfluorooctane sulfonate (PFOS), and testosterone stimulated proliferation in the T98G cells at 500, 1000, and 1000 nM, respectively. However, a perfluorinated salt (ammonium perfluorooctanoate; C8) and a weak androgen (dihydroepiandrosterone; DHEA) did not affect relative cell number in this GBM line, suggesting the proliferative effect is not through the activation of an androgen receptor. Exposure to environmental chemicals that result in a mitogenic response may increase the rate of glioblastoma tumor growth and result in the development of more aggressive forms of GBM tumors.

Influence of estuarine hypoxia on feeding and sound production by two sympatric pipefish species (Syngnathidae).

This research utilizes the acoustic behavior of two sympatric pipefish species to assess the impact of hypoxia on feeding. We collected northern, Syngnathus fuscus, and dusky pipefishes, Syngnathus floridae, from the relatively pristine Chincoteague Bay, Virginia, USA and audiovisually recorded behavior in the laboratory of fish held in normoxic (>5 mg/L O(2)) and hypoxic (2 and 1 mg/L O(2)) conditions. Both species produced high frequency ( approximately 0.9-1.4 kHz), short duration (3-22 msec) clicks. Feeding strikes were significantly correlated with both wet weight of ingested food and click production. Thus, sound production serves as an accurate measure of feeding activity. In hypoxic conditions, reduced food intake corresponded with decreased sound production. Significant declines in both behaviors were evident after 1 day and continued as long as hypoxic conditions were maintained. Interspecific differences in sensitivity were detected. Specifically, S. floridae showed a tendency to perform head snaps at the surface. S. fuscus exhibited a breakdown in the coupling of sound production with food intake in 2 mg/L O(2) with clicks produced in other contexts, particularly choking and food expulsion. Reductions in feeding will ultimately impact growth, health, and eventually reproduction as resources are devoted to survival instead of gamete production and courtship. This work suggests acoustic monitoring of field sites with adverse environmental conditions may reflect changes in feeding behavior in addition to population dispersal.

Changes in the timing of reproduction following chronic exposure to ibuprofen in Japanese medaka, Oryzias latipes.

Effluents from wastewater treatment plants and untreated sewage constitute a low concentration but continuous source of pharmaceutical products to the aquatic environment. One such drug, ibuprofen, is a non-steroidal anti-inflammatory agent that primarily acts through inhibition of cyclooxygenase (COX) activity. Oryzias latipes (Japanese medaka) were exposed for 6 weeks via water to three concentrations of ibuprofen (1-100 microg/L nominal concentrations) and a water control. Reproductive parameters, including frequency of spawning, fecundity, egg size, and rate of fertilization, were measured for each pair of adult medaka following 6 weeks of exposure. Livers homogenates from exposed individuals were assayed for COX activity and whole individuals were histologically examined for tissue damage. Increasing exposure to ibuprofen significantly increased the number of eggs per reproductive event, but decreased the number of spawning events per week. Liver tissue collected from females had less variability in COX activity with increasing concentration of ibuprofen exposure, and tended to have elevated hepatosomatic indices. No pathological damage was evident the in the gills, livers and head kidneys of animals from the highest exposure group. The results of this experiment begin to show that exposure to chronic low levels of ibuprofen alter the pattern of reproduction and may produce sex-specific responses in teleosts.

Differential parental nutrient allocation in two congeneric pipefish species (Syngnathidae: Syngnathus spp.).

Male seahorses and pipefishes of the family Syngnathidae are heralded for their unique brood pouch structures for incubating embryos. There are three general types of brood pouch with increasing complexity: simple ventral gluing, two pouch flaps and a completely sealed sac. The diversity of functional roles within a type in providing nutrition, aeration and protection to offspring is unknown. Here we reveal significant differences in parental nutrient allocation to embryos for two closely related, sympatric pipefishes with similar brood pouch structure. We document differences in embryo attachment, depletion of pouch fluid nutrients over development and egg nutrient partitioning between Syngnathus floridae and Syngnathus fuscus. In S. fuscus, females produce nutritionally poor eggs and the males implant developing embryos in the brood pouch adjacent to blood vessels. A female-biased breeding population was observed, supporting the hypothesis that the cost of male parental care is high in this species. The loose connection between eggs and brood pouch tissues and the appearance of undeveloped eggs and lipid droplets in the pouch of S. floridae males suggest this species utilizes nutrient-rich eggs produced by females as nurse eggs to supplement embryonic development. A balanced sex-ratio for S. floridae further supports more equal parental contribution. This comparison provides evidence of a decline in female gametic investment and reveals the rapid diversification of syngnathid brood pouch function. Our results indicate gross classification of brooding structures into one of the three general pouch types does not predict the energetic investment of males in parental care. But rather, physiological characterization of the relative investment by each sex to offspring is essential to understanding the functional significance of the brood pouch.