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About 70 bats (Nyctalus noctula) spend their winter in an artificial small volume (0,050 cubic meters) winter roost located in the attic of a building. There is a single small exit to the outside directly from the roost. The roost has been cleaned before winter from bat droppings.
Is inhalated ammonia from bat droppings a potential health risk for them?
Is the inhalation of ammonia a possible health problem for bats in an artificial small volume winter roost? - Biology
Bats are the natural host for a variety of dangerous and often lethal viral agents, such as Hendra, Nipah, Marburg, Ebola and the coronavirus that is the etiological agent of the coronavirus disease 2019 (COVID-19). The adaptive evolution of the bats resulted in a balance between host defense and tolerance, which allows them to co-exist with viruses that are highly pathogenic in humans. Other interesting findings about the biology of the bats are the low rate of cancer incidence and the slow advancement of the ageing process. This article provides an overview on the approaches for the use of bats as animal models for the identification of the physiological factors that render apathogenic deadly viruses and for the analysis of the low occurrence of tumors and the slow progression of ageing.
A number of outbreaks of viral-related diseases have been reported in the last decades in different parts of the world, such as the hemorrhagic fevers induced by Marburg and Ebola viruses [1, 2], fatal respiratory or neurological illnesses that are associated with the Hendra virus infection [3], acute respiratory infection and dangerous encephalitis that may derive from the exposure to the Nipah virus [4] and the coronaviruses [5], which have been responsible for the severe acute respiratory syndrome (SARS) in China in February 2003 [6], the Middle East respiratory syndrome (MERS) in Saudi Arabia in 2012 and for the current pandemic of the coronavirus disease 2019 (COVID-19) [7]. The initial COVID-19 cases were reported from Wuhan, China and it rapidly spread to the rest of the World. Covid-19 has caused more than 3 million deaths worldwide, as of April 16th, 2021.
Epidemiological studies indicated that all the above referenced outbreaks were associated with the zoonotic transmission of bat-borne viruses. Studies on the biology of the bats might shed useful insights on the pathogenicity of potentially deadly viruses in humans and, eventually, lead to the development of novel therapeutic approaches for the treatment of harmful viral diseases, along with the prevention of possible future zoonotic spillovers.
Bats are placental mammals that belong to the order of Chiroptera, which, from the Ancient Greek, stands for hand-wing (χείρ – cheir = hand πτερόν – pteron = wing), as the upper forelimbs were adapted through evolution as wings for the flight. Bats have undergone 64 million years of adaptive evolution and constitute more than 20% of the currently existing mammalian species [8]. So far, 1,423 types of bats have been classified, whereas the overall number of known species of mammals is in the range of 6,400. These unique flying mammals are widely distributed around the world, with the exception of some oceanic islands and regions with extreme climate conditions, such as deserts and, naturally, the Arctic and Antarctic Poles.
Bats roost in caves, crevices, hollowed trees, foliage, barns, bridges and even houses and various types of other buildings and they have a fundamental role for a variety of biological functions, which comprise seed dispersal and pollination, fertilization and in keeping insect populations under control.
Some bat species are homothermic, whereas others are heterothermic. Bats can either utilize hibernation, or a diurnal sporadic torpor to save energy [9] and feed on a wide variety of different types of diets, such as insects, nectar, pollen, fruit, fish and blood, as in the case of Desmodus rotundus that is commonly termed bat vampire. Microbats in particular have the sensing powers of echolocation and magnetoreception that allows them to distinguish the polar north from the south [10-13].
Aerial transport constitutes an advantage however, flying is associated with the high consumption of energy [14]. Bats have metabolic rates when they fly that can be in the range of 2.5 to 3 times higher than the metabolic rates of similar size terrestrial mammals [15]. The high demand of energy causes the depletion of 50% stored metabolic energy in a single day. Flying bats utilize 1200 calories per hour [16-18] and, for this reason, they must have a variety of metabolic adaptation and efficient airflow systems during the flight, in order to cope with the high demand of energy, which would otherwise inevitably result in starvation, with consequent death [19]. For instance, their heart rate increases 4 to 5 times and reaches 1,066 beats per minute during flight [18]. Cyclic bradycardic state is a key factor in optimizing the energy conservation by 10% in non-flying times, which can counteract the effects of high heart rates that take place during flight [18].
Interestingly, regardless of the high metabolic rates and small body sizes, bats exhibit lifespans that can be 3.5 higher than non-flying mammals with a comparable body mass [20, 21]. Six types of bats can live for three decades in the wild: Plecotus auratus, Myotis lucifugus, Myotis brandti, Myotis blythii, Rhinolophus ferrumequinum and Pteropus giganteus [22]. One hypothesis for the longevity of bats is related to their hibernation process, during which the metabolic rate is slowed down. It has been observed that hibernating bats live longer than non-hibernating ones [23, 24].
Additional studies are ongoing for the analysis of microRNA molecules in the delay of the effects of ageing in cells and/or tissues of the bats [25], whereas previous reports showed that telomeres do not get shorter with the progression of age in bats, which could be a further mechanism in contrasting the effects of ageing [26, 27].
The low incidence of cancer is another very interesting biological characteristic of the bats [25, 28]. A recent study demonstrated that bat-derived cells are more resistant to genotoxicity than human and mouse cells [28]. The ABC transporter ABCB1 is expressed in considerably high levels in bat cells and removes toxic substances that may ultimately damage the DNA genome [28].
Other important factors that are involved in the low incidence of cancer in bats are related to the expression of particular kinds of microRNA molecules. Studies have shown that microRNAs that act in the context of tumor suppression are upregulated with the progression of age in bats, whereas microRNAs that might promote carcinogenesis are downregulated [25, 29, 30]. Interestingly, the ageing process induces the opposite effects in all other mammalian species, in which the incidence of malignancies increases with age [25, 29, 30].
A remarkable finding about the immunology of the bats is the limited degree of inflammations, when the host immune system responds to infectious agents. Inflammatory responses might otherwise result in the onset of various pathological conditions in humans. The ability of the bats to limit inflammatory reactions to fight infections contributes to the slowing-down of the ageing progression and to the reduction of ageing-related illnesses [31], including cancer [32-35]. For instance, in humans and mice, NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) functions as an intracellular sensor for a broad range of microbial components, endogenous danger signals that trigger inflammation and various environmental irritants. All these factors stimulate the NLRP3 inflammasome, which, in turn, induces the expression of pro-inflammatory cytokines IL-1β and IL-18, along with the gasdermin D-mediated pyroptotic cell death gene [36-38]. The NLRP3 inflammasome may also be implicated in acute myocardial infarction [39]. In contrast to human and murine systems, bats exhibit low levels of NLRP3 activities, even in the presence of high viral loads, as indicated by studies on three distinct RNA viruses: the influenza virus, coronavirus and Melaka virus [31]. Therefore, the avoidance of acute inflammatory reactions is likely to enable the coexistence and survival of bats to viral infections, which, however, render these animals suitable hosts for viruses like coronaviruses, Hendra virus, Nipah virus, Marburg and Ebola viruses.
Coronaviruses have caused the outbreaks of the severe acute respiratory syndrome (SARS) in China in February 2003, the Middle East respiratory syndrome (MERS) epidemic that was reported in Saudi Arabia in 2012 and the recent pandemic of the coronavirus disease 2019 (COVID-19).
Coronaviruses are enveloped single stranded positive sense RNA viruses with a nucleocapsid of helical symmetry and belong to the Orthocoronavirinae subfamily of the Coronaviridae family, which is in the order of Nidovirales [40, 41].
The size of the genome of the coronaviruses may vary from 26 to 32 kb and, therefore, they are the RNA viruses with the largest genome [42]. Club-like spikes protrude from the round-shaped envelope and confer an electron micrograph imaging that resembles the shape of the solar corona, or halo, which provided the typical name coronavirus [43]. Corona is the Latin term for crown. In turn, the Latin word corona derived from the ancient Greek korone. Until 2020, forty-five types of coronaviruses have been identified.
Coronaviruses cause illnesses in humans, birds, cows, pigs and mice. Coronavirus infections mainly affect the respiratory and gastrointestinal tract in humans, whereas only respiratory tract infections have been reported in birds. Human illnesses may vary from the symptoms of the so-called common cold to more severe pathological conditions, which may be lethal, such as in the cases of SARS, MERS and Covid-19. Coronavirus infections set off diarrhea in pigs and cows [44, 45], whereas they induce hepatitis and encephalomyelitis in mice [46, 47].
As anticipated, coronaviruses target the epithelial cells of the respiratory and gastrointestinal tract in humans and they are mainly internalized in form of aerosols, or droplets. However, coronaviruses can also infect humans via fomite, or through an oral-fecal route.
The spikes of the coronaviruses bind to the angiotensin-converting enzyme 2 (ACE2) receptor of the epithelial cells of the respiratory tract in humans [48]. Studies in pigs showed that the transmissible gastroenteritis coronavirus utilizes the alanine aminopeptidase (APN) receptor [49]. The same receptor is utilized by the coronaviruses to infect the epithelial cells of the gastrointestinal tract in humans [50]. Each coronavirus particle carries on average seventy-four spikes on the envelope [51]. The spikes are 20 nm long and consist of an S protein trimer. The S protein contains an S1 and S2 subunits. The resulting trimeric S protein belongs to the class I fusion protein, which allows for the binding to the cellular receptor, which is followed by the membrane fusion between the viral envelope and the cell membrane. The receptor binding domain (RBD) is situated in the subunit S1 on the head of the spike, whereas the S2 subunit constitutes the stem that holds the spike of the viral surface and, following the protease activation, allows for the fusion process of the viral and cellular membranes. The S1 and S2 subunits are not covalently bound [51].
The natural form of transmission of the etiological agent that causes Covid-19 took place initially in rural areas, starting from bats and affecting other wildlife animals. Subsequently, the virus infected farming and livestock animals and, lastly, humans (Figure 1). For instance, camels mediated the transmission of MERS-CoV to humans in Saudi Arabia [52]. Minks are other farming animals that acquired the coronavirus that causes Covid-19 [52]. Humans, in turn, brought the infectious agent from the rural areas to urban settings and then the coronavirus was transmitted to urban animals, including local bats (Figure 1). The human population that was initially affected by the coronavirus infection comprised guano farmers and residents of areas that comprise the natural habitats of the bats. Human migration was ultimately responsible for the transmission of the Covid-19 etiological agent to the urban settings populations (Figure 1).
Ebola and Marburg viruses belong to the Filoviridae family in the order Mononegavirales and have a linear, non-segmented, single-strand negative RNA genome [53]. These two viruses are characterized by high virulence in humans and induce an often-fatal hemorrhagic fever. Ebola virus was initially identified in 1976 [53]. Since then, 20 known outbreaks of Ebola disease have been reported in Africa. In most cases, the outbreaks occurred in rural areas, whereas the outbreak in 2000 affected a semi-urban part of Uganda. A large Ebola virus outbreak occurred in West Africa from 2013 until 2016 and involved both rural and urban regions of three Countries: Guinea, Sierra Leone and Liberia [54-57]. The official reports indicated at least 28,000 cases of Ebola disease, with more than 11,000 deaths. The lethality rate among the reported cases of infection was 62.9% [53].
The most recent Ebola virus outbreaks were observed in May 2018 and in August 2018 in the Democratic Republic of Congo [58, 59]. The May 2018 outbreak approached the city of Mbandaka, which is essential for the transportation system along the Congo river. Fortunately, the outbreak was rapidly contained and only 54 cases of infections with 33 deaths, were reported [58, 59].
The Marburg virus was initially identified in 1967, following hemorrhagic fever outbreaks that took place simultaneously in research laboratories based in Marburg and Frankfurt in Germany and in Belgrade in the former Republic of Yugoslavia, which is currently Serbia. In total, thirty-two people were infected by the Marburg virus and seven died. The outbreak started from imported African green monkeys. The Marburg virus was transmitted to some investigators inside the research facilities and then the virus was passed to family members and to medical personnel. Subsequent studies showed that the natural host for the Marburg virus was the African fruit bat, Rousettus aegyptiacus [60-62].
The analysis of the Rousettus aegyptiacus genome was instrumental in understanding the antiviral immunity of the African fruit bats, which exhibited drastic differences from all other mammal species in terms of genetic arrangements encoding for particular kinds of natural killer cell receptors, MHC class I genes and type I interferons [63]. Basically, the Rousettus aegyptiacus evolution conferred an immune tolerance to viruses. This unique strategy allows bats to coexist with viruses that are highly pathogenic in other mammals and, especially in humans [63].
The Ebola virus contagion among people may happen via direct contact with the blood, or other bodily fluids of infected individuals [64, 65]. Other types of bodily fluids that may carry the virus comprise mucus, saliva, tears, breast milk, feces, urine and semen. So far, there are no reports indicating that the Ebola virus may be transmitted through sweat. The most common route of transmission among humans is via blood, vomit and feces. Ebola virus can infect an individual through the mouth, nose, eyes, abrasions and wounds. Other types of animals that may contract the Ebola virus from the African fruit bats include chimpanzees, gorillas, baboons and duikers, which are a type of antelopes. All these animals may acquire the Ebola virus by eating fruits that were previously bitten by infected African fruit bats [66].
Ebola virus, along with other filoviruses, exhibit high replication rates in several cell types, such as macrophages, monocytes, dendritic cells, hepatocytes, adrenal gland cells and fibroblasts [67]. The Ebola virus replication induces high levels of inflammatory responses, leading to a septic state [68]. The hemorrhagic symptoms appear following the Ebola virus infection of endothelial cells, which occurs within three days after the primary infection of an individual [69].
The membrane of the Ebola and Marburg viruses carries a glycoprotein (GP), which contains two subunits, termed GP1 and GP2. The GP1/2 complex mediates the viral entry into target cells, whereas the soluble GP has a variety of functions, including the inactivation of neutrophils and disfunctions of the vascular system. The soluble GP inactivates neutrophils by binding the CD16b receptor, which, in turn, induces apoptosis in lymphocytes. GP1/2 complex binds C-type lectins, such as L-SIGN, DC-SIGN and hMGL, which are expressed on the membrane of macrophages, monocytes and dendritic cells [70-73]. Another cellular receptor for the Ebola virus is the phosphatidylserine receptor T cell immunoglobulin mucin domain-1 (TIM-1), which is present on the membrane of T lymphocytes [74]. Following the binding to the cellular receptor, the Ebola and Marburg viruses are internalized via endocytosis into the endosome, where the viral membrane fuses with the membrane of the endosome membranes, causing the release of the virion into the cytoplasm [75].
The Hendra and Nipah viruses are enveloped RNA viruses and belong to the genus Henipavirus, in the order Mononegavirales of the family Paramyxoviridae [76].
Sporadic cases of Hendra virus infections were reported among horses between 1994 and 2010 in Brisbane, a suburb of Hendra, in Australia. Rare cases of infections in humans were also reported from 1994 until 2013. The Hendra virus was transmitted to horses by flying foxes, which belong to the genus of megabats [76].
Infected horses develop a strong febrile respiratory illness, often leading either to the death of the animal, or to euthanasia. Two persons that were in contact with the infected animals contracted a flu-like disease. One of them died as a result of an acute severe interstitial pneumonia [76]. The Hendra virus was isolated from a kidney of the deceased patient and characterized. Zoonotic Hendra virus transmission to humans can also cause fatal encephalitis and multi-organ failure. The rate of fatalities among infected humans was 57% [76].
Nipah virus was identified in 1999, when an outbreak affected pigs and humans in Malaysia and Singapore. Almost 300 people were infected and more than 100 patients died. Studies confirmed that the Nipah virus was transmitted by fruit bats [77, 78]. Subsequent Nipah virus outbreaks affected the Indo-Bangladesh regions [79]. Nipah virus infection causes fever, encephalitis and/or respiratory distress in patients [79]. The fatality rate of the Nipah virus has been estimated at 40% to 75% [4].
|Types of bat
|Flying fox
|Silver-haired bat
|Orphaned baby bat
|Formula milk
|Baby bat
|Formula milk
|Captive-born vampire bat
Bats care in research settings is very time consuming and requires sensitivity and dedication from investigators and animal facility technicians. Bats must be able to adapt to the conditions of the artificial situation of a research setting. In their natural environment, most bats procure the food in flight. Obviously, this is no longer possible in a research facility, in which the animals must be individually trained in eating mealworms (Tenebrio), or other types of food from a Petri dish. Each bat may require up to 30 minutes for the initial training feeding period. In some cases, bats learn how to feed independently in three days, whereas in other cases it may take up to four weeks for the animals to learn how to feed themselves from a dish. The links of some videos showing feeding techniques for bats are listed in Table 1. Naturally, the feeding training in a research setting, or in a zoo must take place in the evening, as bats maintain their nocturnal pattern in captivity. It has been estimated that each big brown bat can eat up to 60 mealworms per night. Therefore, large mealworm cultures are required. Other types of bats consume a big quantity of fruit, whereas the co-called vampire bats require blood (Table 1).
Bats can be captured from their roosts in the summer, or from the hibernacula in the winter season. Licenses or permits are required in the majority of countries for fieldwork, capturing and ultimately transportation of animals. In addition, the Centers for Disease Control and Prevention (CDC) has warned about the risk of the transmission of rabies from bats to humans and/or to other animals and has therefore recommended to avoid direct physical contact and to keep the bats in quarantine for a period of three months, which is the standard incubation period for rabies [80, 81]. The quarantined animals have to be monitored by specialized personnels to detect the onset of rabies-related symptoms. Bats must be also inspected for the presence of ectoparasites and fungal infections. Naturally, another important concern is related to the possible transmission of coronaviruses to humans. Studies conducted in the summer of 2006 and 2007 detected group 1 coronaviruses in bats in North America [82] and in Canada [83], respectively.
It is necessary to take all precautions that are required to avoid harm to bats, when they are caught and transported to research facilities. Adult female bats should be captured in the autumn, or winter. If the animals are caught in the summer, the investigators must make sure to release lactating females, to prevent the death by starvation of young bats. Signs of lactation in females can be easily identified by enlarged mammary glands and by the presence of bare skin around the nipples. Capturing hibernating bats can be easily carried out in the winter season. Thousands of hibernating bats can be found in caves and they can be simply taken manually from their hanging position. However, preventive measures must be adopted to minimize as much as possible the awakening of other hibernating bats, which is a process that requires energy. If the animals are disturbed frequently, they will deplete a lot of energy, in a period when the food is not available, which, in turn, may result in death of the animals because of starvation. For this reason, caves should not be visited more than once, or, eventually, twice.
|Type of trapping device
|Notes for the trapping device
|Mist nets
|How to set up a mist net
|How to remove a bat from a mist net
|Harp trap
|How to set up a harp trap
|Using harp traps to capture bats
|youtube youtube
|Wire-mesh cylinder with a smooth, fairly wide metal rim at the upper border
|Link to the document:
During the non-hibernating seasons, bats can be captured with various devices and different strategies. One of the first devices that was developed to capture bats consists of a wire-mesh cylinder with a smooth metal rim situated at the upper border [84]. Other trapping devices are based on mist nets to catch bats as they emerge from a roost [82] and harp traps set up at the opening of a cave during a mating swarm [83]. Table 2 reports the links to the videos, or the descriptions of various trapping devices for the harmless capture of bats.
Motor vehicle transportation of captured bats requires a narrow mesh cage, with a handle on a sliding cover, which can be locked [85]. The wire mesh cage has to be tightly positioned within a wooden box with slits, so the air can circulate. The base of the cage consists of a metal tray, which holds a wet foam rubber sheet for thirsty bats [85].
The rooms for the housing of bats cannot have gaps, through which the animals might eventually escape. Small size bats have the ability to squeeze themselves into openings that are only 1 centimeter wide. This is the typical space that can be found in the perimeter of ventilation shafts screens and drainage systems, or the gap between the floor and the bottom part of a door. Fine wire mesh screens can be utilized to seal off various openings, whereas the bottom part of doors require either rubber, or flexible plastic sheets [86].
Long-term captivity requires different housing conditions for non-hibernating and hibernating bats. Housing is certainly easier for non-hibernating bats, as they only need a standard room that is usually utilized to quarantine the animals after the capture. The temperature of the room for the day and night has to be set on a biweekly basis, in order to mimic the typical outdoor conditions. The same goes for the photoperiod, which has to be adapted to outdoor conditions. The intensity of the light must be kept low in the environment. If there is a window, the room does not need illumination. The humidity of the room is not a critical factor during the non-hibernating period. The typical bat cage for non-hibernating animals is a walk-in wire mesh and is sufficiently large to allow for short flights. Obviously, the door has to be efficiently closed. The dimensions of the bat cage are in the range of 3 x 1 x 2 meters. Smaller cages covered with either a towel, or dark plastic are placed on shelves inside the bat cage. Bats require this type of setting for hiding [86]. It is important that bats are allowed to fly for half an hour inside the holding room, before feeding each day. Urine and feces of the bats will be dropped on paper towels that are present at the bottom of the cages. In this way, it will be easier to remove the waste, which will be then incinerated. It is sufficient to clean the cages once a month, unless the feces are dropped on the wire mesh. In this case, the cages must be cleaned more frequently [86].
Hibernating bats require a dark, humid cold room set at 5C in the winter season, in order to stimulate the hibernation process [86]. The animals must accumulate up to 50% of fat of the body weight, prior to hibernation. The average amount of fat that is utilized by each animal during the state of hibernation is in the range of 1 gram per month. Hibernating bats can be placed in cold rooms inside mouse cages with a wire mesh cover. The typical dimensions of mouse cages are 28 x 20 x 15 centimeters. Not more than three bats can be accommodated inside the same mouse cage. The humidity is an important factor for bats during the hibernation period. In order to keep high humidity, it is necessary to place a stack of 25 wet folded paper towels into the mouse cages. A pad composed of plastic and many layers of cotton has to be placed around the inner walls of the mouse cage and must be in contact with the wet paper towels in the bottom. The cover will keep the pad in the proper position, inside the mouse cage. A coarse dish towel has to be placed underneath the cover, to leave a small space to allow for the circulation of the air. Another stack of wet paper towels is then placed on top of the lid and wrapped with a plastic sheet, leaving a narrow airspace.
A Petri dish with water and one with mealworm larvae must be present in each cage, as bats eventually arouse from hibernation and feel the need to drink, eat, urinate, defecate and re-hibernate. If the experiment requires the removal of bats during the hibernation period, each animal must be alone in the cage, so other hibernating bats will not be disturbed. The cages must be regularly checked every six to eight weeks to restock mealworms and remoisten paper towels.
Breeding of bats in captivity is not practical and it is not recommended. Bats are monestrous animals and their mating in a research setting would require an extensive care. Pregnant females should be collected from the hibernacula, toward the end of the hibernating season. The mating takes place in the autumn and the females store the sperm in the genital tract until the end of the hibernation, when the ovulation and eventually the fertilization occur. The gestation period is comprised between eight to ten weeks. Each pregnant female gives birth to one or two young.
In research facilities, bats can be easily picked up by manual restraint, when the animals are either in the hanging, or sitting position [87]. The wings are immobilized with a gentle pressure from the thumb and the middle finger of the writing hand, while the index finger is pointed to the caudal head region of the animal. The fingers of the opposite hand slide forward under the belly to reach and close the jaw from below, when the animal is gently pulled by the writing hand. At this stage, the bat cannot bite and has to release the claws, as a result of the gentle pull. Following the release of the claws, the bat places the legs on the hand of the operator, which is now bent to enfold the animal. At this juncture, the thumb and the middle finger can be removed from the wings, without squeezing the bat, which is now in a relaxing position [87]. If required by the protocol, the bat can be immobilized on top of a cotton pad placed on the table, by holding the head and the wings, as already described. In the presence of bright light, the eyes of the animal must be loosely covered with a towel, to prevent agitation [87].
The use of anesthetics in bats may be avoided with the use of hypothermia. The animals are placed overnight in a cold room set at 10 C. Vein punctures and other nonpainful procedures may be conducted on animals in a cold-induced torpid state. Painful surgical techniques require the use of anesthetics, in addition to hypothermia. Anesthesia can be administered with an intraperitoneal injection of pentobarbital sodium at doses ranging from 40 to 60 mg per kg of body weight, to induce either light or deep sleep, respectively. The room has to be noise free and the animals cannot be touched until the anesthetic is effective, which may require up to 15 minutes. Following the administration of the anesthetic, the animals must be placed in plastic mouse cages with paper towels in the bottom. Light cotton cloth is used to cover loosely the anesthetized bats. The effect of the anesthetic wears down roughly one-hour post-injection. The recovery time is approximately one hour at a temperature of 23 C, or more than three hours at 20 C. Animals must have access to water placed in a Petri dish. However, the bats must fast overnight, in order to prevent vomiting.
Blood collection from bats requires a stereoscopic microscope for the puncturing of the blood vessels. The total volume of blood that can be collected is in the range of 10% of the lean body weight of a brown bat. A cotton pad must be placed underneath the bat, which has to be gently restrained with a gloved hand, with the fingers slightly bent [87]. The torpid bat settles in this position and falls asleep. The thumb of the restraining hand is moved aside, while the other hand spreads the wing with caution and holds it down gently against the cotton pad with the index finger. A light source pointed at the wing will show the vessels and intensify the circulation of the blood. A hypodermic needle can be utilized to puncture a small vessel and to collect the blood sample.
A flexible cannula is required for the collection of blood from arteries or veins. A 4 cm PE 10 polyethylene tubing is attached to a 30-gauge hypodermic needle, lacking a luer lock hub, whereas the other end of the tubing is connected to a Tuohy-Borst Adapter, which, in turn, is inserted into a hypodermic syringe [87]. If large quantities of blood are required, the bat has to be euthanized by cervical dislocation [88], the thoracic cavity must be immediately opened and the blood is withdrawn from the heart. In addition to cervical dislocation, bats can be euthanized by decapitation, or by an overdose of anesthetic.
Urine samples can be obtained from bats in the awakening period, which is the normal time when bats urinate. Therefore, the operators must remove the bats from the cage while they are still sleeping, hold them in an upright position and place a small glass vial under the eternal urethral opening. The vial has to be encased by masking tape to provide support to the hind legs of the bats. Fecal samples can be easily taken from the paper towels that have been placed in the bottom of the cage. For fresh feces samples, the collection has to be carried out as the animals feed, because bats frequently defecate while they eat.
Some bat-derived cell lines are currently available from the American Tissue Culture Collection (ATCC) (10801 University Boulevard, Manassas, VA 20110 USA) and Millipore Sigma (PO Box 14508, St. Louis, MO 63178, USA) (Table 3).
|Organism, age and gender
|Culture properties
|Culture medium
|Catalog number
|Tadarida brasiliensis, bat, free-tailed. Adult, female
|Dulbecco's Modified Eagle's Medium, 10% heat-inactivated fetal bovine serum and 1% L-glutamine (2 mM stock solution)
|Tb 1 Lu (ATCC® CCL-88™)
|Tadarida brasiliensis, bat, free-tailed. Adult, female
|Dulbecco's Modified Eagle's Medium, 10% heat-inactivated fetal bovine serum and 1% L-glutamine (2 mM stock solution)
|Myotis velifer incautus, bat, mouse -eared. Adult, female
|Skin-derived tumor
|Epithelial tumor
|Dulbecco's Modified Eagle's Medium, 10% heat-inactivated fetal bovine serum and 1% L-glutamine (2 mM stock solution)
|Myotis velifer incautus, bat, mouse-eared. Adult, female
|Interscapular tumor
|Epithelial tumor
|Dulbecco's Modified Eagle's Medium, 10% heat-inactivated fetal bovine serum and 1% L-glutamine (2 mM stock solution)
|Macrotus waterhousi californicus, bat, mouse-eared. Age and gender not specified
|Unknown. Possibly heart
|This cell line is neither produced nor fully characterized by ATCC.
|Not specified.
|Dulbecco's Modified Eagle's Medium, 10% heat-inactivated fetal bovine serum and 1% L-glutamine (2 mM stock solution)
|Original organism not specified. Adult, female
|EMEM (EBSS), + 10% Fetal Bovine Serum (FBS), 1% L-Glutamine (2 mM stock solution), 1% Non-essential amino acids (NEAA)
|Millipore Sigma
|TB1 Lu (NBL-12)
Several other bat-derived cell lines have been produced by various research laboratories (Table 4) [89-92]. Bat fetuses can also be utilized for the production of cell lines and they might be available from local zoos (Table 4) [91].
|Organism, age and gender
|Cell line name
|Culture properties
|Culture medium
|Title and bibliographic reference (PMID)
|Pipistrellus ceylonicus. (Vespertilionidae). Embryo. Female
|Dulbecco's Modified Eagle's Medium (DMEM), with 10% heat inactivated fetal bovine serum and 1% L-glutamine (200 mM stock solution).
|Establishment of cell line from embryonic tissue of Pipistrellus ceylonicus bat species from India & its susceptibility to different viruses [89]
|Perimyotis subflavus (tricolored bat). Adult. Gender not specified
|Dulbecco's modified Eagle medium (DMEM)/F12-Ham's media, with 15% heat-inactivated fetal bovine serum, 1% non-essential amino acids (NEAA) and 1% L-glutamine (200 mM stock solution).
|Evidence supporting a zoonotic origin of human coronavirus strain NL63 [90]
|Rousettus aegyptiacus (Egyptian fruit bat). Fetus. Gender not specified
|Head of the fetus Body of the fetus Vertebrate column of the fetus
|Ro5T Ro6E Ro5R
|Dulbecco's modified Eagle medium (DMEM)/F12-Ham's media, with 5% gamma irradiated fetal calf serum and 1% L-glutamine (200 mM stock solution).
|Cell lines from the Egyptian fruit bat are permissive for modified vaccinia Ankara [91]
|Epomops buettikoferi (Büttikofer's epauletted fruit bat)
|Dulbecco's Modified Eagle's Medium (DMEM), with 10% heat inactivated fetal bovine serum, 1% L-glutamine (200 mM stock solution), 1% sodium pyruvate (100 mM stock solution) and 1% MEM non-essential amino acids (NEAA) (100 X stock solution).
|Type I Interferon Reaction to Viral Infection in Interferon-Competent, Immortalized Cell Lines from the African Fruit Bat Eidolon helvum [92]
|Eidolon helvum (African fruit bat). Adult. Gender not specified
|Dulbecco's Modified Eagle's Medium (DMEM), with 10% heat inactivated fetal bovine serum, 1% L-glutamine (200 mM stock solution), 1% sodium pyruvate (100 mM stock solution) and 1% MEM non-essential amino acids (NEAA) (100 X stock solution).
|Type I Interferon Reaction to Viral Infection in Interferon-Competent, Immortalized Cell Lines from the African Fruit Bat Eidolon helvum [92]
|Rousettus aegyptiacus (Egyptian fruit bat). Adult. Gender not specified
|Dulbecco's Modified Eagle's Medium (DMEM), with 10% heat inactivated fetal bovine serum, 1% L-glutamine (200 mM stock solution), 1% sodium pyruvate (100 mM stock solution) and 1% MEM non-essential amino acids (NEAA) (100 X stock solution).
|Type I Interferon Reaction to Viral Infection in Interferon-Competent, Immortalized Cell Lines from the African Fruit Bat Eidolon helvum [92]
|Myotis daubentoniid (Vespertilionidae). Adult. Gender not specified
|Dulbecco's Modified Eagle's Medium (DMEM), with 10% heat inactivated fetal bovine serum, 1% L-glutamine (200 mM stock solution), 1% sodium pyruvate (100 mM stock solution) and 1% MEM non-essential amino acids (NEAA) (100 X stock solution).
|Type I Interferon Reaction to Viral Infection in Interferon-Competent, Immortalized Cell Lines from the African Fruit Bat Eidolon helvum [92]
Bats have recently emerged as interesting animal models for the study of human diseases and ageing. A particular emphasis has been placed on the coronaviruses that have been responsible for the current Covid-19 pandemic and for the previous pandemics termed SARS and MERS. Studies can also be conducted for other viral agents, such as Ebola and Marburg viruses, Hendra virus and Nipah virus.
The biology of the bats is revealing distinct characteristics from other mammalian species, in terms of immune responses and slow progression of the ageing process. Studies have shown that immune responses and ageing process in bats seem to rely on different patterns of genetic and epigenetic expression that typically occur in all the other mammalian species [35].
Bats cannot be bred in the context of animal facilities. However, capturing bats require special precautions, in order to prevent the transmission of harmful infectious agents to the operators. Lastly, the feeding of the animals necessitates a particular dedication from the researchers, especially in the initial period, when the bats must be trained to feed from a Petri dish. On one hand, the lack of a syngeneic bat model can be perceived as a drawback. On the other hand, however, the type of studies on the immunology, slow progression of the ageing process and low incidence of cancer in bats can only be conducted on wild-type animals, albeit in captivity. Of course, bats must be carefully classified in the attempt to standardize as much as possible the results among different investigational settings. Disparities in the physiology of the bats is most probably going to affect the experimental outcomes. On these grounds, it is essential to use the same type of bats for all the experimental procedures to achieve consistent and reproducible findings.
In Europe, artificial bat roosts have long been tested, particularly in silviculture (Bäumler, 1988 Issel & Issel, 1955 Natuschke, 1960 Schwenke, 1983) which led to a more successful design of artificial roost boxes (Schwenke, 1983). More recently, the value of insectivorous bats for agriculture and the use of bat houses in agricultural landscapes have received growing attention (e.g., Boyles et al., 2011 Puig-Montserrat et al., 2015 Taylor et al., 2018). Nevertheless, an ever growing human population and related land use changes, especially agricultural intensification, have led to a threat of extinction to about one quarter of global bat species (see Figure 1.3 in Voigt & Kingston, 2016 Mickleburgh, Hutson & Racey, 2002 Tilman et al., 2001 Tscharntke et al., 2012). The loss of roost sites caused by land use changes is one of the major drivers of this decline (Mickleburgh, Hutson & Racey, 2002 Park, 2015) and there is a particular lack of knowledge regarding roost site preferences of African bat species (Monadjem et al., 2009 Monadjem et al., 2010a Taylor, 2000). Given accelerating land use change from natural to agricultural landscapes in the developing world and an assumed decline of South African bat populations (Voigt & Kingston, 2016), proactive management of these populations is indispensable to sustain the ecosystem services they provide (Cumming et al., 2014 Taylor et al., 2017 Tuttle, Kiser & Kiser, 2013). Proactive management of bat populations requires filling knowledge gaps about roost site preferences for African bat species, in particular around intensive agricultural systems (Monadjem et al., 2009 Park, 2015 Taylor, 2000).
Peer-reviewed studies focusing on artificial roost site use by African bat species are non-existent and most studies have been conducted in Europe, followed by North America and Australia (Rueegger, 2016). Summarizing these, bat species seem to have a general preference for large volume and multiple compartment bat houses mounted on poles or houses rather than on trees (Rueegger, 2016). There also seems to be a preference for bat houses built from woodcement although these studies are mostly from Europe (Dodds & Bilston, 2013 Gerell, 1985 Haensel & Tismer, 1999 Rueegger et al., in press). Generally, the microclimate of bat houses (influenced by e.g., insulation, sun exposure and color) seems to be an important factor for bat house occupancy (Fukui et al., 2010 Rueegger, 2016 Shek et al., 2012). Looking at different designs and colors of bat houses, studies suggest that preferences also vary greatly depending on the reproductive state of females (Baranauskas, 2009 Fukui et al., 2010 Flaquer, Torre & Ruiz-Jarillo, 2006 Kerth, Weissmann & König, 2001). Furthermore, many bat species seem to be sensitive to competition for bat houses by other species including birds, social bees, ants and wasps (Baranauskas, 2009 Dodds & Bilston, 2013 Meddings et al., 2011). There is a need for research on artificial roost site use, especially in Africa, and this is the first peer-reviewed study of bat house occupancy in South Africa. Successful bat house design and deployment seem to relate mostly to the climatic region and bat species targeted (Rueegger, 2016). Therefore, the objective of this study was to gain insight into the preference for different artificial roost sites by insectivorous bats in macadamia orchards in South Africa. The main research question was: What are the key features of occupied artificial roost sites? We hypothesize that bat houses providing the warmest microclimate will have higher occupancy.
During 122 days, from mid-May to mid-September 2017, we recorded 2928 temperature data points for each of the 18 Classic 4-chamber bat boxes, which were either mounted on poles (n = 6), non-heated buildings (n = 6), or heated buildings (n = 6) at two sites in Quebec, Canada. Based on a generalized additive mixed model, we estimated that average daily temperatures of Classic bat boxes varied among mounting types, sites, and with time of day (Fig. 1, Supplementary Tables S2 and S3). At night, the Classic bat boxes average temperature was between 1 and 1.5 °C warmer when mounted on heated or non-heated buildings than poles while the opposite occurred during the day (Fig. 1). The percentage of time below, between, and above the EOTR were relatively similar among mounting type. Temperature varied from 6.5 to 44 °C in Classic bat boxes on poles, from 6.5 to 48.5 °C in Classic bat boxes on non-heated buildings, and from 7 to 49 °C in Classic bat boxes on heated buildings. The minimal and maximal temperature of Classic bat boxes were generally similar among mountings at the same site, varying from 0.5 to 5 °C (Table 1).
Estimated hourly patterns for the different Classic bat box mountings in Quebec, in 2017 (pole: n = 6, non-heated building: n = 6, and heated building: n = 6). The estimated values are based on a generalized additive mixed model, accounting for time, date, orientation, external temperature, site, and individual bat box identity. Values of fixed factors have been set to: date = July 6, orientation = east, external temperature = 18 °C. The asterisks (*) represent significant differences between structures during the day and night. P = pole and B = building.
During 122 days, from mid-May to mid-September 2017, we recorded 2928 temperature data points for each of the 18 Classic bat boxes, which were either facing east (n = 4), south (n = 4), and west (n = 4) on buildings, or facing east (n = 2), south (n = 2) and south-east (n = 2) on poles, at two sites in Quebec, Canada. Based on a generalized additive mixed model, we estimated that average daily temperatures of Classic bat boxes varied among orientations, mounting types, sites, and with time of day (Fig. 2, Supplementary Tables S4 and S5). On buildings, west-facing Classic bat boxes were significantly warmer in the early evening and colder in the early morning than those facing east. During the day, temperatures in east-facing Classic bat boxes were significantly different than those facing south or west, being warmer especially in the morning (Fig. 2). On poles, the only significant difference was at mid day, when south-facing Classic boxes were warmer than those facing east. The percentage of time below, between, and above the EOTR varied among orientations. The highest percentage of time in between the EOTR occurred for Classic bat boxes with an easterly orientation, while the highest percentage of time above the EOTR occurred for bat boxes with an east and west orientation at the warmer site. Temperature varied from 6.5 to 49 °C in Classic bat boxes facing east, from 6.5 to 44 °C in Classic bat boxes facing south-east, 6.5–41 °C in Classic bat boxes facing south, and from 6.5 to 49 °C in Classic bat boxes facing west. The minimal and maximal temperature of Classic bat boxes were generally similar, among orientations at the same site, varying from 0.5 to 5 °C (Table 1).
Estimated hourly patterns for the different bat box orientations in Quebec, in 2017. Bat boxes facing east (n = 4), south (n = 4), and west (n = 4) on buildings (a), and facing east (n = 2), south (n = 2) and south-east (n = 2) on poles (b). The estimated values are based on a generalized additive mixed model, accounting for time, date, external temperature, structure, site, and individual bat box identity. Values of fixed factors have been set to: date = July 6, external temperature = 18 °C. The asterisks (*) represent significant differences between orientations during the day and night. E = east, S = south, W = west, and SE = south-east. HB = heated building.
From mid-May to mid-September 2016–2019, we recorded 2928 temperature data points per year and per bat box at seven sites in Quebec, Canada. We tested in total 12 bat box designs mounted on poles and buildings facing east, but only present results comparing the thermodynamics of our best newly designed model, the Ncube PH1, which included a main and a lower chamber (n = 8), to the Classic 4-chamber (n = 11). Based on a generalized additive mixed model, we estimated that average daily temperatures varied between the Classic and the main chamber of the Ncube PH1 model, (Fig. 3, Supplementary Tables S6 and S7, see Supplementary Fig. S1 for all models). Temperatures in the Classic and Ncube PH1 were significantly warmer than the outside temperature during both night and day. The main chamber of the Ncube PH1 was significantly warmer than the Classic during both night and day, being on average 3.5 ± 1.5 °C warmer during the night, 3 ± 1.5 °C warmer in afternoon, and similar in the morning from 700 to 1100.
Estimated hourly patterns for the Classic (n = 11) and the Ncube PH1 models (main and lower chambers, n = 8). The estimated values are based on a generalized additive mixed model accounting for time, week, year, structure, external temperature, site, and individual bat box identity. Values of fixed factors have been set to: week = first half of July, year = 2019, structure = building, external temperature = 18 °C. The asterisks (*) represent significant differences between models during the day and night.
Bat boxes were installed at seven sites separated into cooler (n = 2, Tx̅ in June = 11 °C), intermediate (n = 3, Tx̅ in June = 16 °C), and warmer sites (n = 2, Tx̅ in June = 19 °C). For comparative purposes, we present the percentage of time below, in between, and above the EOTR for bat boxes on buildings at intermediate sites only results at warmer and cooler sites being similar with a higher and lower mean temperature respectively (Table 2, see Supplementary Fig. S2 for all models). The Classic was in the EOTR 46% of the time and above the EOTR 2% of the time. Considering the bats can use the lower chamber when above 40 °C in the main chamber, the Ncube PH1 was in the EOTR 58% of the time and never above the EOTR, therefore increasing by 12% the amount of time in the EOTR compared to the Classic.
The minimal temperature of the Classic and the Ncube PH1 were similar (2 °C and 1.5 °C respectively) but the maximal temperature differed given the use of the lower chamber of the Ncube PH1 (48 °C and 40 °C respectively Table 2). From mid-May to mid-September 2019, daily minimum temperatures recorded in the Classic were slightly warmer than external temperatures, but slightly colder than the Ncube PH1 main chamber at both cooler 1 and warmer 2 sites (Fig. 4). Daily maximum temperatures in the Classic were warmer than external temperatures, similar to the Ncube PH1 lower chamber, and colder than the Ncube PH1 main chamber at the site cooler 1. At the site warmer 2, daily maximum temperatures in the Classic were warmer than the Ncube PH1 lower chamber, but similar to the Ncube PH1 main chamber, both overheating frequently, with 50 and 54 days with daily maximum temperatures over 40 °C respectively (Fig. 4). Overheating events occurred only once at warmer sites for the Ncube PH1 lower chamber on buildings, which spent < 1% of the time above 40 °C (one two hours long overheating bout Table 2). Overheating events occurred at warmer and intermediate sites for the Classic model, which spent 4% of the time above 40 °C at warmer sites (52 1–6 h long overheating bouts in 2019) and 2% at intermediate sites (12 1–6 h long overheating bouts in 2019).
Daily minimum and maximum temperatures in the Classic and the Ncube PH1 bat boxes on buildings and the external temperature in 2019 at cooler and warmer sites, in Quebec. (a) daily maximum temperatures at the cooler site, (b) daily maximum temperatures at the warmer site, (c) daily minimum temperatures at the cooler site, (d) daily minimum temperatures at the warmer site. The red dotted lines represent the extended optimal temperature range of 22–40 °C.
Bioenergetic modeling
We estimated average daily thermoregulatory energy expenditures using bioenergetic modeling for a female little brown bat during the gestation and lactation period based on internal temperatures recorded in 2019 in a Classic 4-chamber versus a Ncube PH1 bat box on building at cooler, intermediate, and warmer sites in Quebec, Canada. We selected the internal temperature of the Ncube PH1 main chamber when equal or lower than 40 °C and the lower chamber when above 40 °C. The internal temperature of the Classic was based on the middle chamber (chamber 3 from the front) at all times (see the methods for more details). Predicted average daily energy expenditure was reduced by 3–8% during gestation in the Ncube PH1 compared to the Classic bat box model (Fig. 5). During lactation, energy savings varied between 5 and 7%. Energy saving differences were higher in cooler sites compared to warmer sites but were significant at all sites and reproductive periods, except during gestation at warmer sites.
Average daily thermoregulatory energy expenditure (in kilojoules) from bioenergetic modeling for a female bat during the gestation and lactation period in a Classic versus Ncube PH1 bat box on building at: (a) cooler, (b) intermediate, and (c) warmer sites in Quebec, in 2019.
General responses
My analysis of the literature included results of studies of bat responses to silvicultural treatments conducted on over 70 species across three continents. By grouping all studies within treatments, using broad response categories (positive, negative, neutral), and grouping species into guilds, some of the subtleties of species-specific responses to variations in treatments (e.g., fire severity) and variations in study design (e.g., detector placement, time since treatment) were lost. Nonetheless, some general patterns regarding the effects of silvicultural treatments on temperate insectivorous bat foraging and roosting behavior emerged and generally were consistent with the conclusions of Hayes and Loeb (2007) and Law et al. (2016), who conducted more traditional literature reviews. Negative responses to silvicultural treatments were more common in studies examining roosting behavior than foraging behavior, suggesting that bat foraging and commuting habitat use may be less affected by changes in forest structure and composition than roost habitat use ( Figs. 2 and 3). In addition, among both roosting and foraging studies, bats were more likely to show positive or neutral responses to mid-rotation treatments that reduced clutter while retaining overstory structure such as thinning and prescribed fire compared to treatments that removed all or most of the overstory.
As predicted, edge- and open-space foragers showed negative responses to regrowth forest both for foraging and roosting habitat. Prior to self-thinning or mechanical thinning, regrowth forests usually have a dense canopy and subcanopy resulting in a high degree of clutter ( Guldin et al. 2007). Most studies that documented negative responses to regrowth forests attributed low activity or use to the dense vegetation found in these forests ( Erickson and West 1996 Parker et al. 1996 Humes et al. 1999 Law and Chidel 2001, 2002 Adams et al. 2009 Law et al. 2019). Thinning regrowth stands often increases foraging activity ( Humes et al. 1999 Blakey et al. 2016 Gonsalves et al. 2018a, 2018b) leading to the hypothesis that regrowth forests are avoided because of dense clutter. A further hypothesis is that regrowth forests provide poor roosting habitat because of the scarcity of large snags for roosting ( Erickson and West 1996 Parker et al. 1996 Humes et al. 1999), and dense vegetation that reduces solar radiation necessary for passive rewarming and prevents easy access to and egress from roosts ( Kunz and Lumsden 2003 Barclay and Kurta 2007).
Although not statistically significant, foraging activity of closed-space foragers tended to decrease in response to clearcut and shelterwood harvests in most studies and did not recover in young regrowth forests, although neutral responses were observed in some studies. In contrast, open-space foragers showed a strong positive foraging response to clearcut or shelterwood harvest treatments and edge-space foragers also tended to respond in a positive or neutral fashion to harvesting. Because many species, particularly edge-space foragers, are more likely to forage near edges of cuts than in the center, some of the variation in responses by closed- and edge-space foragers to clearcut and shelterwood harvests may have been due to detector placement in acoustic studies ( Grindal and Brigham 1999 Hogberg et al. 2002 Law and Law 2011 Webala et al. 2011) harvest tract size also may have been a factor ( Grindal and Brigham 1998). Open-space foragers avoid highly cluttered habitats ( Aldridge and Rautenbach 1987 Fenton 1990) therefore, it is not surprising that many studies documented increases in foraging and commuting activity or occupancy in response to the creation of open areas through harvesting for this group. In addition to creating open habitats with reduced clutter, harvesting results in hard edges between forest and open areas, thus generating habitat for both edge- and open-space foragers. Edges between forest and open areas provide important foraging and commuting areas for many species of bats because they aid in navigation, provide protection from predators, and have greater amounts of insect prey ( Verboom et al. 1999 Law and Law 2011 Kalcounis-Rueppell et al. 2013).
Due to minimal tree overstory in areas that have undergone clearcut or shelterwood harvests, it is not surprising that both closed-space and edge- and open-space foragers showed predominantly negative roosting responses to these harvesting treatments. Nevertheless, western long-eared bats (Myotis evotis) in British Columbia sometimes use remnant stumps after clearcutting, possibly because of the thermal advantages associated with full solar exposure in an open habitat ( Vonhof and Barclay 1997). Thus, even though regeneration harvests such as clearcutting and shelterwood harvests result in the loss of roosting habitat for bats of most species, a few bats are able to take advantage of the remaining structures.
Plantation forests often lack structures commonly used by forest bats for roosting, such as snags, hollow trees, and hardwood trees with large canopies ( Ruczyński et al. 2010 Burgar et al. 2015). While bats use these forests for foraging, particularly edge- and open-space foragers ( Fig. 2), studies to date indicate that they avoid roosting in these forests ( Fig. 3). Nonetheless, these forests represent important habitat for some species such as the endangered New Zealand long-tailed bat and lesser short-tailed bat (Mystacina tuberculata rhyacobia). Both species were assumed to rely on native forest but have since been recorded roosting and foraging in plantation forests ( Borkin and Parsons 2010a, 2010b). Thus, highly managed forests such as plantations should not be dismissed as bat habitat ( Russo et al. 2010).
The creation of gaps within intact forests often resulted in positive foraging responses by edge- and open-space foragers. Similar to clearcuts, gaps provide uncluttered foraging space as well as a hard edge ( Crome and Richards 1988 Menzel et al. 2002 Loeb and O'Keefe 2011 Ketzler et al. 2018). It also has been hypothesized that higher insect abundance in gaps compared to closed-canopy forests contributes to greater use by foraging bats ( Tibbels and Kurta 2003). Although I found no studies that explicitly tested the effects of forest gaps on roost use or selection, many studies have found that bats of some species commonly select roosts on the edges of, or near, gaps ( Campbell et al. 1996 Callahan et al. 1997 Carter and Feldhamer 2005 Fabianek et al. 2015). Many studies have hypothesized that roosting on the edge of canopy gaps may allow bats to decrease energetic costs because of high solar radiation in these sites as well as close access to foraging sites ( Loeb and O'Keefe 2011).
Responses of foraging bats to thinning and fire were similar ( Fig. 2). In general, the response to thinning and fire by foraging bats was neutral or positive, especially for edge- and open-space foragers. Positive responses may be due to changes in forest structure, insect prey availability, or a combination of the two. Both thinning and fire reduce clutter ( Peterson and Reich 2001 Fulé et al. 2004 Phillips et al. 2004), and observed positive foraging responses have often been attributed to a reduction of clutter ( Smith and Gehrt 2010 Armitage and Ober 2012 Inkster-Draper et al. 2013 Gonsalves et al. 2018a). Studies that simultaneously examined insect availability and bat activity in relation to thinning found no significant effects of thinning on insect biomass, community composition, or abundance leading to the conclusion that changes in bat activity post-thinning were in response to structural changes in the habitat ( Tibbels and Kurta 2003 Morris et al. 2010 Blakey et al. 2016 Gonsalves et al. 2018a, 2018b).
Although bats' responses to thinning appear to be due to structural changes, whether bats respond to changes in forest structure or insect abundance postfire is not clear. For example, Armitage and Ober (2012) and Cox et al. (2016) reported no increase in insect abundance in burned stands compared to unburned stands even though they found an increase in bat use of burned stands. In contrast, Lacki et al. (2009) and Malison and Baxter (2010) reported that use of foraging areas or bat activity was related to increases in insect abundance after prescribed or wildland fire and Nyctophilus geoffroyi in Australia were 1.1 g heavier and had shorter torpor bouts and longer normothermic bouts immediately following a wildfire compared to 2 years postfire ( Doty et al. 2016). Doty et al. (2016) hypothesized that the physiological responses of N. geoffroyi were due to greater prey abundance immediately after the fire as well as easier detection of prey due to reduced ground cover. Understanding responses of insect communities to fire and subsequent responses of bat communities may be confounded by differences among studies in burn severity ( Malison and Baxter 2010 Burns et al. 2019), fire extent ( Law et al. 2018b), time since last burn ( Doty et al. 2016), fire frequency ( Law et al. 2019), and the insect communities ( Perry 2012). Thus, it is not possible at present to isolate which factor or factors bats are responding to postfire.
The number of studies examining the effects of thinning and fire on roost habitat use (n = 19) was far lower than the number of studies examining these treatments on foraging habitat use (n = 34), most likely leading to the nonsignificant results for close-space foragers. Nonetheless, some patterns still were evident. As predicted, closed-space foragers in several studies increased their use of, or selected, burned areas for roosting, but they tended to avoid thinned areas for roosting ( Fig. 3). Thinning may provide some of the structural characteristics of good roost habitat (e.g., reduced clutter resulting in increased solar radiation and easier access and egress to roosts) but thinned stands may be too young in some cases to contain appropriate roost structures such as large snags, large trees with cracks and crevices, or large-canopy hardwoods or conifers for foliage roosters ( Kalcounis-Rueppell et al. 2005 Barclay and Kurta 2007 Carter and Menzel 2007). Thinning also may reduce the number of snags in the stand ( Law et al. 2016, 2018c). In contrast, prescribed fires as well as wildfires often occur in mature or old-age stands which contain larger roost structures than younger stands. Fire also may create suitable roost structures such as snags and cracks and crevices in live trees ( Burns 1955 Paulsell 1957 Boyles and Aubrey 2006) although the net gain of snags is primarily in the small-size classes ( Horton and Mannan 1988). However, in some cases, fires destroy snags or hollow-bearing trees ( Parnaby et al. 2010 Lindenmayer et al. 2012). Thus, while mechanical thinning likely will increase foraging habitat for some bat species, fire appears to be more effective at creating both roosting and foraging habitat.
Several caveats regarding the studies included in this review should be considered when evaluating the preceding results and when designing studies in the future. These include: 1) the categorization of bats into foraging guilds 2) reliance on acoustic detectors to study treatment effects on foraging habitat use and the assumptions inherent in these types of studies 3) the strong bias toward conducting studies primarily during summer and 4) the assumption that greater use of a particular treatment results in higher population levels or viability.
Many ways exist to categorize bats into foraging guilds based on wing morphology, body size, and echolocation call structures (e.g., Aldridge and Rautenbach 1987 Fenton 1990 Schnitzler and Kalko 2001) and these categories vary based on particular bat communities and on the researchers who define them. For example, some studies included in this review categorized species both by morphology and echolocation call structure (e.g., Adams et al. 2009), requiring me to combine categories used in their studies. The same species also may be placed in different guilds by different researchers. Menzel et al. (2005), for instance, classified big brown bats (Eptesicus fuscus) as open-space foragers, whereas Jantzen and Fenton (2013) categorized them as edge-space foragers. While I was consistent in classifying species across studies based on the majority of authors' categorizations when data were presented for individual species, this was not always possible when data were grouped into guilds. Thus, some species may have been included in more than one foraging guild in some analyses in this review. As with any categorization of biological entities, it is likely that many species lie somewhere on a continuum among categories and thus, use of different categories or characterizations of species than those used here may result in slightly different conclusions.
While use of acoustic detectors has greatly increased researchers' abilities to study bat habitat use and bat responses to silvicultural treatments and other natural and anthropogenic disturbances, several things must be considered when designing acoustic studies and interpreting data ( Britzke et al. 2013). In addition to issues related to species identification (e.g., Broders et al. 2004 Lemen et al. 2015 Russo and Voight 2016), using acoustic detectors to understand habitat use has many assumptions ( Gannon et al. 2003). Hayes (2000) outlined practical solutions for dealing with some of these assumptions such as calibrating detectors to correct for variation among detectors and appropriate study designs to deal with temporal variation. These solutions eliminate or decrease differences in detectability across time and among similar habitats but, they ignore differences in detectability related to treatment effects, particularly if those treatments result in variation in clutter ( Patriquin et al. 2003). For example, Burns et al. (2019) found that detection probabilities of Eptesicus fuscus–Lasionycteris noctivagans and Lasiurus borealis–Nycticeius humeralis were significantly higher in areas that had been burned compared to unburned sites. If treatment effects on detection probability are not included in the analyses of use or activity, then conclusions about the effects of silvicultural treatments on bat activity or occupancy may be biased ( MacKenzie 2005). Of the 55 studies that used acoustic detectors to examine treatment effects on bat activity or occupancy, only Burns et al. (2019) and Starbuck et al. (2015) accounted for detection probability in their analyses of treatment effects. Thus, results of studies that use acoustic detectors and do not consider treatment effects on detection probability in their analyses may be biased. Inclusion of detection probabilities in future studies will result in more robust results.
Only 19 of the 88 studies (22%) considered in this review included data from outside the summer maternity season. While understanding how silvicultural treatments affect bats during the maternity season is very important, many critical life history stages occur during other seasons, including breeding, migration, preparation for hibernation, and hibernation itself ( Weller et al. 2009). Migration is energetically expensive and although long-distance migrants such as L. noctivagans use heterothermy to reduce the need to stop and feed along the migratory route ( McGuire et al. 2014), they require appropriate roost structures for use of torpor during the day. In addition, bats that enter hibernation with greater fat stores are more likely to survive white-nose syndrome ( Cheng et al. 2019) suggesting that providing high-quality foraging habitat during fall is critical for species affected by this disease. Treatments such as prescribed fire often occur during fall, winter, and early spring and if studies are restricted to summer only, the direct or immediate effects of these treatments on bats are ignored ( Braun de Torrez et al. 2018). In addition, clutter may be considerably reduced in deciduous forests during fall, winter, and early spring after leaf fall which may result in different patterns of use among silviculturally treated and untreated sites compared to summer. Other factors such as roost microclimates and insect abundances also differ among seasons ( Kerth et al. 2001 Leksono et al. 2005 Ruegger 2019), which may affect relative use in treated and untreated sites.
Another assumption of most studies included in this review is that high activity levels or selection of a habitat for foraging or roosting equates to good habitat that ultimately will result in high abundance or viable populations ( Barclay and Kurta 2007). High population density may not indicate better quality habitat but instead represent a habitat sink ( Van Horne 1983 Pulliam 1988). Because bats have high mobility, changes in relative habitat use simply may reflect a shift in use from one treatment type to another but not reflect changes in populations. In addition, high prey abundance may mean that bats need less time to obtain required resources, resulting in lower activity levels. I only was able to find one study that examined demographic responses of temperate-zone bats to forest management ( Law et al. 2018a). In contrast to most studies, which found negative effects of regrowth forest on bat foraging and roosting habitat use, Law et al. (2018a) found minimal effects of regrowth forest on abundance and survival of four species of bats over a 14-year period. As suggested by Law et al. (2016), long-term population studies are required to understand the full impact of silvicultural treatments on bats.
Examining physiological responses of bats to disturbance caused by silvicultural treatments is another way to determine their effects on bat demographics and viability ( Geiser et al. 2018 Stawski and Doty 2019). The study by Doty et al. (2016), however, was the only study in this review to examine the physiological consequences of bats to any treatment. Physiological responses of N. geoffroyi (greater body mass, shorter torpor durations, and longer normothermic bout durations) immediately after a wildfire compared to 2 years postfire generally support studies based on foraging responses and suggest that bats tend to respond positively to fire. In contrast, in a tropical forest subject to logging, body weight and leukocyte numbers of some bat species were lower in areas recently disturbed by logging compared to unlogged areas ( Seltmann et al. 2017). Results of these few studies suggest that to understand fully the effects of silvicultural treatments on bat populations and fitness, researchers need to measure parameters such as survival, reproductive success, or the physiological consequences of using one treated area versus another, as well as relative use and selection.
Future research needs and directions
Researchers have made considerable advances in understanding the effects of forest practices on bats over the past 40 years, but a great deal still needs to be learned. Several hypotheses need to be tested regarding forest management effects on bats at the population and landscape levels to allow managers to implement management strategies that provide healthy forests, needed goods and services, and roosting and foraging habitat for bats. For example, testing the hypothesis that the potential creation of roosts and decrease in clutter resulting from prescribed fire is a more favorable treatment for bats than thinning will help inform decisions regarding the best fuel reduction methods (e.g., Waldrop et al. 2016). Similarly, the hypothesis that plantation forests are avoided because they lack suitable roosts is important for industrial foresters and could be tested experimentally by providing artificial roosts. Several hypotheses that examine the effects of fire parameters on bats and their insect prey also need to be tested, including the hypothesis that bats respond differently to fires of different severities, time since last burn, frequencies, season, and extent. Testing the hypothesis that bats respond differently to prescribed fires and wildfires is important to understanding the importance of the use of prescribed fire as a tool for preventing destructive wildfires ( Fernandes and Botelho 2003).
Hypotheses regarding study designs and their effects on the results of forest management research studies also need to be tested. For instance, researchers need to test the hypothesis that results gained from acoustic studies are similar to those gained from tracking studies when testing foraging responses to silvicultural treatments. Further, the lack of research on silvicultural treatments outside the reproductive period highlights the need to test the hypothesis that treatment effects will vary across seasons and species or guilds. Finally, studies are needed to test the hypothesis that bats will respond to silvicultural treatments at the population level (i.e., with increases or decreases in demographic parameters such as survival and reproductive success).
This review focused on studies undertaken at the stand scale and most studies were carried out over short time periods. But forest management occurs at the landscape scale ( Guldin et al. 2007) and considering how treatments affect bat communities across large landscapes and over long time periods is important. For example, although edge- and open-space foraging bats often responded positively to clearcutting and shelterwood harvests, these areas eventually become regrowth forest, to which bats generally showed negative responses. Thus, many studies call for a mix of silvicultural treatments and forest age and composition classes across the forest (see Law et al. 2016). Effective forest management that aims to conserve or to recover bat populations will need to determine the level and timing of harvesting necessary to provide sufficient early, mid-, and late rotation forests to satisfy the roosting and foraging needs of all bats in the community over time. Spatially explicit landscape models such as LANDIS and LANDIS II, which simulate future forest conditions across a large landscape based on various forest management scenarios (e.g., amount, timing, and spatial distribution of harvests and other forest management practices), have been used to simulate availability of habitat over time for wildlife, including bats ( Shifley et al. 2006 Pauli et al. 2015). Spatial distribution models coupled with outputs of forest management or disturbance scenarios may also produce fruitful insights (e.g., Bosso et al. 2018). Factors such as connectivity among suitable habitats also need to be considered ( Henderson and Broders 2008 Farrow and Broders 2011 Frey-Ehrenbold et al. 2013). Results of the studies reviewed here and of future studies can be used to develop long-term landscape models that can be used for effective bat conservation across large landscapes and long time scales.
Multispecies, Multipathogen Dynamics
Very few studies in any wildlife system have adequately described either multihost pathogen dynamics or multipathogen dynamics in a single host species. This is hardly surprising given the complexities involved in understanding single pathogen–single host dynamics. However, there are important studies that suggest both multihost and pathogen dynamics are important in other non-bat systems, and empirical data that suggest these situations may occur in bat infection systems. Through analysis of time series data, Telfer et al. (2010) demonstrated statistically that in a parasite community, including a virus, protozoan and two bacteria, within individual field voles (Microtus agrestis), risk of infection was altered by concurrent infection to a greater extent than by age or season. Lello et al. (2004 ) demonstrated that, in a rabbit (Oryctolagus cuniculus) population, gut helminth community parasites either compete or exist in mutualistic relationships. Few studies have considered multiple infections in bats ( Drexler et al., 2011 ). Muhldorfer et al. (2011) detected infections in 12% of 486 bats from 19 European bat species, detecting co-infection with herpesviruses in five bats, but were unable to infer much from this study as it was based on opportunistic sampling. Also, in Thailand, two distinct clades of Nipah virus were found to circulate in the same colony of the fruit bat, Pteropus lylei ( Wacharapluesadee et al., 2010 Rahman et al., 2010 )
From a multihost community perspective, Davies and Pedersen (2008) found that host-relatedness and geographical range overlap were significant predictors of pathogen sharing among primates. Some viruses, notably RABV, are promiscuous, infecting multiple host species. Streicker et al. (2010) demonstrated that although RABV variants predominantly circulate within single host species, they are able to spill over into other species. Similar to Davies and Pedersen (2008) , Streicker et al. (2010) documented highly asymmetrical patterns of cross-species RABV transmission in the North American bat fauna, with host-relatedness and geographical range overlap being the strongest predictors of cross-species transmission, thus also suggesting an important influence of host sympatry. Whether these factors affect cross-species transmission of other lyssaviruses within Chiroptera, or from bats to other mammals, is not known, because isolations are often few and serological findings may be due to cross-reactivity between related species or variants, complicating interpretation (e.g. Wright et al., 2010 ). European bat lyssaviruses 1 and 2 (EBLV-1, EBLV-2) appear to show a very narrow host range in Europe. EBLV-1 circulates in Serotine bats (Eptesicus serotinus) and EBLV-2 in Daubenton's bat (Myotis daubentonii) this host fidelity is, however, not complete, as the first isolation of EBLV-2 was from a Pond bat (Myotis dasycneme), and in Spain, EBLV-1 sequences have been recovered from several bat species ( Serra-Cobo et al., 2002 Amengual et al., 2007 ). The importance of cross-species transmission events in seasonally changing communities may vary with respect to pathogen or variants of a pathogen, but is not really clear for any system. In some cases, infection cycles may be maintained in co-roosting species without cross-species transmission. For example, Kuzmin et al. (2011) discovered that Commerson's leaf-nosed bat (Hipposideros commersoni) is a possible reservoir of Shimoni bat virus (SHIBV, a lyssavirus), while Egyptian fruit bats (Rousettus aegyptiacus) and Miniopterus spp. bats in the same caves were seropositive against Lagos bat virus (LBV) and West Caucasian bat virus (WCBV) ( Kuzmin et al., 2008b ), thus suggesting that at least for these lyssaviruses, infections may circulate among specific host species and transmission may be minimal among sympatric bats. Moreover, Cui et al. (2007) reported clustering of CoV sequences from geographically separated Vespertilionid bats of the same species, even for co-roosting bats. Coronaviruses from Rhinolophidae bats, however, did not share this feature and appear to have undergone a number of host shifts.
Future directions for research to address the role of multiple hosts in infection dynamics and multiple pathogens in infection dynamics
Research programmes that focus on multiparasite and/or multihost systems, following the approach described previously, will help advance our understanding of the ecology of bat diseases. Particular care is needed to consider the markedly reduced statistical power available when considering the dynamics of co-infections, and modelling to help plan empirical data collection would be particularly beneficial for such studies ( Restif et al., in press ). These studies could particularly benefit from community ecology approaches, testing for inter-specific interactions, such as described by Telfer et al. (2010) . Additionally, more detailed molecular techniques and robust co-evolutionary studies could be incorporated that tease out cross-species transmission events (e.g. Cui et al., 2007 Streicker et al., 2010 ) and infection dynamics (e.g. Drexler et al., 2011 ).
Foraging and habitat use
Studies of migration have comprised the main use of radar in entomology and ornithology, but radar techniques have also been applied to studies of non-migratory 'station keeping' behaviours, such as foraging (in the wide sense of appetitive movements to find the resources required for survival, somatic growth and reproduction Dingle 2014 ). The application of radar to insect foraging is reviewed in Drake and Reynolds (2012, chapter 14). The main technology employed in these studies has been harmonic radar (Drake and Reynolds 2012, chapter 8 Kissling et al. 2014 ), where a transponder is attached to the individual to be tracked. This device returns signals to the radar at twice the transmitted frequency and, because the receiver is selectively tuned to this shifted frequency, all unwanted radar reflections ('clutter') from ground features (which would normally obscure a low-flying insect target) are suppressed. There are two forms of entomological unit using the harmonic principle: azimuthally-scanning 'true' radars which provide geometrically accurate maps of the insects' flight trajectories, and harmonic direction-finders – portable instruments which do not provide range information but allow the operator to move in the direction from which the strongest signals are received, and thus home in on a tagged insect much as one might using traditional radio tracking.
Azimuthally-scanning harmonic radar is particularly suited to bee tracking studies, and this technology has already furthered work on bee navigation and pollinator ecology (see references in Drake and Reynolds 2012, Lihoreau et al. 2012 and Woodgate et al. 2016 ), as well as documenting the (deleterious) effects of sub-lethal doses of neonicotinoid insecticides and glyphosate herbicide on bee foraging behaviour and navigational performance (Fischer et al. 2014 , Balbuena et al. 2015 , Tison et al. 2016 ). These studies form part of the accumulating evidence of harm to wild bees and honeybees by neonicotinoids in the environment and will have assisted in informing the probable ban on all uses of these insecticides on outdoor crops. There have also been scanning harmonic radar studies of foraging and short-range dispersal in butterflies (Cant et al. 2005 , Ovaskainen et al. 2008 ), indicating that this technique can be applied to taxa other than bees (see also Kissling et al. 2014 ).
Harmonic direction-finders have been used mainly on studies of pest insects, but also on some beneficial species such as natural enemies of pests (e.g. carabid beetles) and on other (non-insect) invertebrate species of conservation concern (see references in chapter 14 of Drake and Reynolds 2012, Kissling et al. 2014 ). The study species often disperse by terrestrial locomotion or, if they can fly, do so mainly over short distances only. Useful information on the localized flight of more mobile species (e.g. dragonflies Hardersen 2007) has also been obtained by this technology. Allied techniques such as radio telemetry have also been employed on insects large enough to carry active transmitters (reviewed by Kissling et al. 2014 ). A typical example might be the monitoring of short-range dispersal flights of an endangered scarabaeid beetle Osmoderma eremita which lives in old hollow trees (Hedin et al. 2008 , Svensson et al. 2011 ).
In contrast to entomology, radar has rarely been applied to study foraging or other local movements of individual birds and mammals. Active radar transponders were used to trace coyotes Canis latrans and further developed to be used e.g. in seabirds (French and Priede 1992 ). At least under calm weather conditions, birds interacting with fishing vessels (Fig. 6, Assali et al. 2017 ) or bats foraging over the open sea (Ahlén et al. 2009 ) can be observed by marine radar. Many seabird species feed in large numbers at fishing vessels on offal or bait, where their high mortality is the main threat to populations worldwide (Phillips et al. 2016 ). But the extent of overlap and behaviour in relation to ships is poorly known (Fig. 6). Using novel biologging devices, which detect radar emissions and record the position of boats and seabirds, Weimerskirch et al. ( 2018 ) measured the extent of the overlap between albatrosses and fishing vessels and generated estimates of the intensity of fishing and distribution of vessels in international waters, which has widespread implications for bycatch risk in seabirds and identification of areas of intense fishing throughout the ocean.
Tracks of gulls approaching and foraging at a fishing vessel (upper right corner) as visualized by an off-the-shelf X-band marine radar (25 kW peak power) installed on a research platform in the southern North Sea (distance between rings: 1 nautical mile). Herring gulls Larus argentatus, great black-backed gulls Larus marinus and other gulls use the helicopter deck of the platform for resting and to 'wait' for fisheries activities to feed on discards and offal (Hüppop et al. 2008 ).
Analysis of WSR data has revealed that densities of waterbirds increased in response to temporary wetland habitat established for migrating birds, following the Deepwater Horizon oil spill in the Gulf of Mexico (Sieges et al. 2014 ). Similarly, densities of waterfowl as measured by WSR increased in response to restoration of wetland habitat as part of the Wetland Reserve Program in the U.S. (Buler et al. 2010). In both of these examples, access to archived WSR data proved critical to establishing baseline measures of bird use prior to habitat alteration.
Hazards of pesticides
Direct impact on humans
If the credits of pesticides include enhanced economic potential in terms of increased production of food and fibre, and amelioration of vector-borne diseases, then their debits have resulted in serious health implications to man and his environment. There is now overwhelming evidence that some of these chemicals do pose a potential risk to humans and other life forms and unwanted side effects to the environment (Forget, 1993 Igbedioh, 1991 Jeyaratnam, 1981). No segment of the population is completely protected against exposure to pesticides and the potentially serious health effects, though a disproportionate burden, is shouldered by the people of developing countries and by high risk groups in each country (WHO, 1990). The world-wide deaths and chronic diseases due to pesticide poisoning number about 1 million per year (Environews Forum, 1999).
The high risk groups exposed to pesticides include production workers, formulators, sprayers, mixers, loaders and agricultural farm workers. During manufacture and formulation, the possibility of hazards may be higher because the processes involved are not risk free. In industrial settings, workers are at increased risk since they handle various toxic chemicals including pesticides, raw materials, toxic solvents and inert carriers.
OC compounds could pollute the tissues of virtually every life form on the earth, the air, the lakes and the oceans, the fishes that live in them and the birds that feed on the fishes (Hurley et al., 1998). The US National Academy of Sciences stated that the DDT metabolite DDE causes eggshell thinning and that the bald eagle population in the United States declined primarily because of exposure to DDT and its metabolites (Liroff, 2000). Certain environmental chemicals, including pesticides termed as endocrine disruptors, are known to elicit their adverse effects by mimicking or antagonising natural hormones in the body and it has been postulated that their long-term, low-dose exposure is increasingly linked to human health effects such as immune suppression, hormone disruption, diminished intelligence, reproductive abnormalities and cancer (Brouwer et al., 1999 Crisp et al., 1998 Hurley et al., 1998)
A study on workers (N=356) in four units manufacturing HCH in India revealed neurological symptoms (21%) which were related to the intensity of exposure (Nigam et al., 1993). The magnitude of the toxicity risk involved in the spraying of methomyl, a carbamate insecticide, in field conditions was assessed by the National Institute of Occupational Health (NIOH) (Saiyed et al., 1992). Significant changes were noticed in the ECG, the serum LDH levels, and cholinesterase (ChE) activities in the spraymen, indicating cardiotoxic effects of methomyl. Observations confined to health surveillance in male formulators engaged in production of dust and liquid formulations of various pesticides (malathion, methyl parathion, DDT and lindane) in industrial settings of the unorganised sector revealed a high occurrence of generalised symptoms (headache, nausea, vomiting, fatigue, irritation of skin and eyes) besides psychological, neurological, cardiorespiratory and gastrointestinal symptoms coupled with low plasma ChE activity (Gupta et al., 1984).
Data on reproductive toxicity were collected from 1,106 couples when the males were associated with the spraying of pesticides (OC, OP and carbamates) in cotton fields (Rupa et al., 1991).A study in malaria spraymen was initiated to evaluate the effects of a short-term (16 week) exposure in workers (N=216) spraying HCH in field conditions (Gupta et al., 1982).
A study on those affected in the Seveso diaster of 1976 in Italy during the production of 2,4,5 T, a herbicide, concluded that chloracne (nearly 200 cases with a definite exposure dependence) was the only effect established with certainty as a result of dioxin formation (Pier et al., 1998). Early health investigations including liver function, immune function, neurologic impairment, and reproductive effects yielded inconclusive results. An excess mortality from cardiovascular and respiratory diseases was uncovered, possibly related to the psychosocial consequences of the accident in addition to the chemical contamination. An excess of diabetes cases was also found. Results of cancer incidence and mortality follow-up showed an increased occurrence of cancer of the gastrointestinal sites and of the lymphatic and haematopoietic tissue. Results cannot be viewed as conclusive, however, because of various limitations: few individual exposure data, short latency period, and small population size for certain cancer types. A similar study in 2001 observed no increase in all-cause and all-cancer mortality. However, the results support the notion that dioxin is carcinogenic to humans and corroborate the hypotheses of its association with cardiovascular- and endocrine-related effects (Pier et al., 2001). During the Vietnam War, United States military forces sprayed nearly 19 million gallons of herbicide on approximately 3.6 million acres of Vietnamese and Laotian land to remove forest cover, destroy crops, and clear vegetation from the perimeters of US bases. This effort, known as Operation Ranch Hand, lasted from 1962 to 1971. Various herbicide formulations were used, but most were mixtures of the phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Approximately 3 million Americans served in the armed forces in Vietnam during the Vietnam War. Some of them (as well as some Vietnamese combatants and civilians, and members of the armed forces of other nations) were exposed to defoliant mixtures, including Agent Orange. There was evidence on cancer risk of Vietnam veterans, workers occupationally exposed to herbicides or dioxins (since dioxins contaminated the herbicide mixtures used in Vietnam), and of the Vietnamese population (Frumkin, 2003).
Impact through food commodities
For determining the extent of pesticide contamination in the food stuffs, programs entitled 'Monitoring of Pesticide Residues in Products of Plant Origin in the European Union' started to be established in the European Union since 1996. In 1996, seven pesticides (acephate, chlopyriphos, chlopyriphos-methyl, methamidophos, iprodione, procymidone and chlorothalonil) and two groups of pesticides (benomyl group and maneb group, i.e. dithiocarbamates) were analysed in apples, tomatoes, lettuce, strawberries and grapes. An average of about 9 700 samples has been analysed for each pesticide or pesticide group. For each pesticide or pesticide group, 5.2% of the samples were found to contain residues and 0.31% had residues higher than the respective MRL for that specific pesticide. Lettuce was the crop with the highest number of positive results, with residue levels exceeding the MRLs more frequently than in any of the other crops investigated. The highest value found in 1996 was for a compound of the maneb group in lettuce which corresponded to a mancozeb residue of 118 mg/kg. In 1997, 13 pesticides (acephate, carbendazin, chlorothalonil, chlopyriphos, DDT, diazinon, endosulfan, methamidophos, iprodione, metalaxyl, methidathion, thiabendazole, triazophos) were assessed in five commodities (mandarins, pears, bananas,beans, and potatoes). Some 6 000 samples were analysed. Residues of chlorpyriphos exceeded MRLs most often (0.24%), followed by methamidophos (0.18%), and iprodione (0.13%). With regard to the commodities investigated, around 34% contained pesticide residues at or below the MRL, and 1% contained residues at levels above the MRL. In mandarins, pesticide residues were most frequently found at levels at or below the MRL (69%), followed by bananas (51%), pears (28%), beans (21%) and potatoes (9%). MRLs were exceeded most often in beans (1.9%), followed by mandarins (1.8%), pears (1.3%), and bananas and potatoes (0.5%). Estimation of the dietary intake of pesticide residues (based on the 90th percentile) from the above-mentioned commodities, where the highest residue levels of the respective pesticides were found, shows that there is no exceeding of the ADI with all the pesticides and commodities studied (European Commission, 1999). In 1998, four commodities (oranges, peaches, carrots, spinach) were analysed for 20 pesticides (acephate, benomyl group, chlopyriphos, chlopyriphos-methyl, deltamethrin, maneb group, diazinon, endosulfan, methamidophos, iprodione, metalaxyl, methidathion, thiabendazole, triazophos, permethrin, vinclozolin, lambdacyalothrin, pirimiphos-methyl, mercabam). With regard to all four commodities investigated in 1998 (oranges, peaches, carrots, spinach), about 32% contained residues of pesticides at or below MRL, and 2% above the MRL (1.8% for EU-MRLs, 0.4% for national MRLs). Residues at or below the MRL were found most often in oranges (67%), followed by peaches (21%), carrots (11%) and spinach (5%). MRL values were exceeded most often in spinach (7.3%), followed by peaches (1.6%), carrots (1.2%)and oranges (0.7%). The intake of pesticide residues has not exceeded the ADI in any case. It was found to be below 10% of the ADI for all pesticides. The exposure ranges from 0.35% of the ADI for the benomyl group to 9.9% of the ADI for the methidathion group. In 1999, four commodities (cauliflower, peppers, wheat grains, and melon) were analysed for the same 20 pesticides as in the 1998 study (European Commission, 2001). Overall, around 4700 samples were analysed. Residues of methamidophos exceeded MRLs most often (8.7%), followed by the maneb group (1.1%), thiabendazole (0.57%), acephate (0.41%) and the benomyl group (0.35%). The MRL for methamidophos was exceeded most often in peppers and melons (18.7 and 3.7%, respectively). The residues of the maneb group exceeded the MRL most often in cauliflower (3.9%) residues of thiabendazole exceeded the MRL most often in melons (2.8% of the melon samples). With regard to all the commodities investigated, around 22% of samples contained residues of pesticides at or below the MRL and 8.7% above the MRL. Residues at or below MRL were found most often in melons (32%), followed by peppers (24%), wheat grains (21%) and cauliflower (17%). MRL values were exceeded most often in peppers (19%), followed by melons (6.1%), cauliflower (3%) and wheat grains (0.5%). The intake of pesticide residues did not exceed the ADI in any case. It was below 1.5% of the ADI for all pesticides. The exposure ranged between 0.43% of the ADI for methamidophos and 1.4% of the ADI for endosulfan. The intakes for the highest residue levels in a composite sample for chlorpyriphos, deltamethrin, endosulfan and methidathion were below the ARfD for adults. They range between 1.5% of the ARfD for deltamethrin and 67% of the ARfD for endosulfan (Nasreddine and Parent-Massin, 2002). In spite of food contamination, most pesticide deaths recorded in hospital surveys are the result of self-poisoning (Eddleston, 2000). The Global Burden of Disease Study 6 estimated that 798 000 people died from deliberate self-harm in 1990, over 75% of whom were from developing countries (Murray and Lopez, 1996). More recent WHO estimates showed that over 500 000 people died from self-harm in Southeast Asia and the Western Pacific during 2000 alone (WHO, 2001). Suicide is the commonest cause of death in young Chinese women and Sri Lankan men and women (Murray and Lopez, 1996 Sri Lankan Ministry of Health, 1995 WHO, 2001).
In India the first report of poisoning due to pesticides was from Kerala in 1958, where over 100 people died after consuming wheat flour contaminated with parathion (Karunakaran, 1958). This prompted the Special Committee on Harmful Effects of Pesticides constituted by the ICAR to focus attention on the problem (Report of the Special Committee of ICAR, 1972). In a multi-centric study to assess the pesticide residues in selected food commodities collected from different states of the country (Surveillance of Food Contaminants in India, 1993), DDT residues were found in about 82% of the 2205 samples of bovine milk collected from 12 states. About 37% of the samples contained DDT residues above the tolerance limit of 0.05 mg/kg (whole milk basis). The highest level of DDT residues found was 2.2 mg/kg. The proportion of the samples with residues above the tolerance limit was highest in Maharastra (74%), followed by Gujarat (70%), Andhra Pradesh (57%), Himachal Pradesh (56%), and Punjab (51%). In the remaining states, this proportion was less than 10%. Data on 186 samples of 20 commercial brands of infants formulae showed the presence of residues of DDT and HCH isomers in about 70 and 94% of the samples with their maximum level of 4.3 and 5.7 mg/kg (fat basis) respectively. Measurement of chemicals in the total diet provides the best estimates of human exposure and of the potential risk. The risk of consumers may then be evaluated by comparison with toxicologically acceptable intake levels. The average total DDT and BHC consumed by an adult were 19.24 mg/day and 77.15 mg/day respectively (Kashyap et al., 1994). Fatty food was the main source of these contaminants. In another study, the average daily intake of HCH and DDT by Indians was reported to be 115 and 48 mg per person respectively, which were higher than those observed in most of the developed countries (Kannan et al., 1992).
Impact on environment
Pesticides can contaminate soil, water, turf, and other vegetation. In addition to killing insects or weeds, pesticides can be toxic to a host of other organisms including birds, fish, beneficial insects, and non-target plants. Insecticides are generally the most acutely toxic class of pesticides, but herbicides can also pose risks to non-target organisms.
Surface water contamination
Pesticides can reach surface water through runoff from treated plants and soil. Contamination of water by pesticides is widespread. The results of a comprehensive set of studies done by the U.S. Geological Survey (USGS) on major river basins across the country in the early to mid- 90s yielded startling results. More than 90 percent of water and fish samples from all streams contained one, or more often, several pesticides (Kole et al 2001). Pesticides were found in all samples from major rivers with mixed agricultural and urban land use influences and 99 percent of samples of urban streams (Bortleson and Davis, 1987). The USGS also found that concentrations of insecticides in urban streams commonly exceeded guidelines for protection of aquatic life (U.S. Geological Survey, 1999). Twenty-three pesticides were detected in waterways in the Puget Sound Basin, including 17 herbicides. According to USGS, more pesticides were detected in urban streams than in agricultural streams (US Department of the Interior, 1995). The herbicides 2,4-D, diuron, and prometon, and the insecticides chlorpyrifos and diazinon, all commonly used by urban homeowners and school districts, were among the 21 pesticides detected most often in surface and ground water across the nation (U.S. Geological Survey, 1998). Trifluralin and 2,4-D were found in water samples collected in 19 out of the 20 river basins studied (Bevans et al., 1998 Fenelon et al., 1998 Levings et al., 1998 Wall et al., 1998). The USGS also found that concentrations of insecticides in urban streams commonly exceeded guidelines for protection of aquatic life (U.S. Geological Survey, 1999). According to USGS, "in general more pesticides were detected in urban streams than in agricultural streams", (Bortleson and Davis, 1987). The herbicide 2,4-D was the most commonly found pesticide, detected in 12 out of 13 streams. The insecticide diazinon, and the weed-killers dichlobenil, diuron, triclopyr, and glyphosate were detected also in Puget Sound basin streams. Both diazinon and diuron were found at levels exceeding concentrations recommended by the National Academy of Sciences for the protection of aquatic life (Bortleson and Davis, 1987).
Ground water contamination
Groundwater pollution due to pesticides is a worldwide problem. According to the USGS, at least 143 different pesticides and 21 transformation products have been found in ground water, including pesticides from every major chemical class. Over the past two decades, detections have been found in the ground water of more than 43 states (Waskom, 1994). During one survey in India, 58% of drinking water samples drawn from various hand pumps and wells around Bhopal were contaminated with Organo Chlorine pesticides above the EPA standards (Kole and Bagchi, 1995). Once ground water is polluted with toxic chemicals, it may take many years for the contamination to dissipate or be cleaned up. Cleanup may also be very costly and complex, if not impossible (Waskom 1994 O'Neil, 1998 US EPA, 2001).
Soil contamination
A large number of transformation products (TPs) from a wide range of pesticides have been documented (Barcelo' and Hennion, 1997 Roberts, 1998 Roberts and Hutson, 1999). Not many of all possible pesticide TPs have been monitored in soil, showing that there is a pressing need for more studies in this field. Persistency and movement of these pesticides and their TPs are determined by some parameters, such as water solubility, soil-sorption constant (Koc), the octanol/water partition coefficient (Kow), and half-life in soil (DT50). Pesticides and TPs could be grouped into:(a) Hydrophobic, persistent, and bioaccumulable pesticides that are strongly bound to soil. Pesticides that exhibit such behavior include the organochlorine DDT, endosulfan, endrin, heptachlor, lindane and their TPs. Most of them are now banned in agriculture but their residues are still present. (b) Polar pesticides are represented mainly by herbicides but they include also carbamates, fungicides and some organophosphorus insecticide TPs. They can be moved from soil by runoff and leaching, thereby constituting a problem for the supply of drinking water to the population. The most researched pesticide TPs in soil are undoubtedly those from herbicides. Several metabolic pathways have been suggested, involving transformation through hydrolysis, methylation, and ring cleavage that produce several toxic phenolic compounds. The pesticides and their TPs are retained by soils to different degrees, depending on the interactions between soil and pesticide properties. The most influential soil characteristic is the organic matter content. The larger the organic matter content, the greater the adsorption of pesticides and TPs. The capacity of the soil to hold positively charged ions in an exchangeable form is important with paraquat and other pesticides that are positively charged. Strong mineral acid is required for extracting these chemicals, without any analytical improvement or study reported in recent years. Soil pH is also of some importance. Adsorption increases with decreasing soil pH for ionizable pesticides (e.g. 2,4-D,2,4,5-T, picloram, and atrazine) (Andreu and Pico', 2004).
Effect on soil fertility (beneficial soil microorganisms)
Heavy treatment of soil with pesticides can cause populations of beneficial soil microorganisms to decline. According to the soil scientist Dr. Elaine Ingham, "If we lose both bacteria and fungi, then the soil degrades. Overuse of chemical fertilizers and pesticides have effects on the soil organisms that are similar to human overuse of antibiotics. Indiscriminate use of chemicals might work for a few years, but after awhile, there aren't enough beneficial soil organisms to hold onto the nutrients" (Savonen, 1997). For example, plants depend on a variety of soil microorganisms to transform atmospheric nitrogen into nitrates, which plants can use. Common landscape herbicides disrupt this process: triclopyr inhibits soil bacteria that transform ammonia into nitrite (Pell et al., 1998) glyphosate reduces the growth and activity of free-living nitrogen-fixing bacteria in soil (Santos and Flores, 1995) and 2,4-D reduces nitrogen fixation by the bacteria that live on the roots of bean plants (Arias and Fabra, 1993 Fabra et al., 1997), reduces the growth and activity of nitrogen-fixing blue-green algae (Singh and Singh, 1989 Tözüm-౺lgan and Sivaci-Güner, 1993), and inhibits the transformation of ammonia into nitrates by soil bacteria (Frankenberger et al., 1991, Martens and Bremner, 1993). Mycorrhizal fungi grow with the roots of many plants and aid in nutrient uptake. These fungi can also be damaged by herbicides in the soil. One study found that oryzalin and trifluralin both inhibited the growth of certain species of mycorrhizal fungi (Kelley and South, 1978). Roundup has been shown to be toxic to mycorrhizal fungi in laboratory studies, and some damaging effects were seen at concentrations lower than those found in soil following typical applications (Chakravarty and Sidhu, 1987 Estok et al., 1989). Triclopyr was also found to be toxic to several species of mycorrhizal fungi (Chakravarty and Sidhu, 1987) and oxadiazon reduced the number of mycorrhizal fungal spores (Moorman, 1989).
Contamination of air, soil, and non-target vegetation
Pesticide sprays can directly hit non-target vegetation, or can drift or volatilize from the treated area and contaminate air, soil, and non-target plants. Some pesticide drift occurs during every application, even from ground equipment (Glotfelty and Schomburg, 1989). Drift can account for a loss of 2 to 25% of the chemical being applied, which can spread over a distance of a few yards to several hundred miles. As much as 80% of an applied pesticide can be volatilised within a few days of application (Majewski, 1995). Despite the fact that only limited research has been done on the topic, studies consistently find pesticide residues in air. According to the USGS, pesticides have been detected in the atmosphere in all sampled areas of the USA (Savonen, 1997). Nearly every pesticide investigated has been detected in rain, air, fog, or snow across the nation at different times of the year (U.S. Geological Survey, 1999). Many pesticides have been detected in air at more than half the sites sampled nationwide. Herbicides are designed to kill plants, so it is not surprising that they can injure or kill desirable species if they are applied directly to such plants, or if they drift or volatilise onto them. Many ester-formulation herbicides have been shown to volatilise off treated plants with vapors sufficient to cause severe damage to other plants (Straathoff, 1986). In addition to killing non-target plants outright, pesticide exposure can cause sublethal effects on plants. Phenoxy herbicides, including 2,4-D, can injure nearby trees and shrubs if they drift or volatilise onto leaves (Dreistadt et al., 1994). Exposure to the herbicide glyphosate can severely reduce seed quality (Locke et al., 1995). It can also increase the susceptibility of certain plants to disease (Brammall and Higgins, 1998). This poses a special threat to endangered plant species. The U.S. Fish and Wildlife Service has recognized 74 endangered plants that may be threatened by glyphosate alone (U.S. EPA Office of Pesticides and Toxic Substances, 1986). Exposure to the herbicide clopyralid can reduce yields in potato plants (Lucas and Lobb, 1987). EPA calculated that volatilisation of only 1% of applied clopyralid is enough to damage non-target plants (US EPA, 1990). Some insecticides and fungicides can also damage plants (Dreistadt et al., 1994). Pesticide damage to plants is commonly reported to state agencies in the Northwest. (Oregon Dept. of Agriculture, 1999 Washington Dept. of Health, 1999). Plants can also suffer indirect consequences of pesticide applications when harm is done to soil microorganisms and beneficial insects. Pesticides including those of new the generation, e.g., dacthal, chlorothalonil, chlorpyrifos, metolachlor, terbufos and trifluralin have been detected in Arctic environmental samples (air, fog, water, snow) (Rice and Cherniak, 1997), and (Garbarino et al., 2002). Other studies have identified the ability of some of these compounds to undergo short-range atmospheric transport (Muir et al., 2004) to ecologically sensitive regions such as the Chesapeake Bay and the Sierra Nevada mountains (LeNoir et al., 1999 McConnell et al., 1997 Harman-Fetcho et al., 2000, Thurman and Cromwell , 2000). One long-term study that investigated pesticides in the atmosphere of British Columbia (BC), dating from 1996 (Belzer et al., 1998) showed that 57 chemicals were investigated at two sampling sites (Agassiz and Abbotsford) in the Fraser Valley, from February 1996 until March 1997. Atrazine, malathion, and diazinon, highly toxic chemicals identified as high-priority pesticides by Verrin et al. (2004), were detected as early as the end of February (72 pg/m 3 ) until mid-October (253 pg/m 3 ), with a peak concentration in mid-June of 42.7 ngm 𢄣 . Dichlorvos is a decomposition product of another pesticide, Naled (Dibrom) (Hall et al., 1997). Captan and 2,4-D showed the highest concentrations and deposition rates at these two sites, followed by dichlorvos and diazinon (Dosman and Cockcraft, 1989). Air concentrations of currently used pesticides in Alberta were investigated in 1999 at four sampling sites that were chosen according to geography and pesticide sales data (Kumar, 2001). Triallate and trifluralin were the two mostly detected pesticides at the four sites. Insecticides (malathion, chlorpyrifos, diazinon and endosulfan) were detected intermittently with concentrations in the range 20 pg/m 3 . South of Regina, Saskatchewan, in 1989 and 1990, 2,4-D reached 3.9 and 3.6 ng/m 3 at the end of June (Waite et al., 2002a). Triallate, dicamba, bromoxynil concentrations were also higher in 1989 (peak concentration of 4.2 ng/m 3 in mid-June) compared with 1990 (600 pg/m 3 in mid-June). In a more recent study, Waite et al. (2005) studied spatial variations of selected herbicides on a threesite, 500km transect that included two agricultural sites𠅋ratt's Lake, located 35 km southwest of Regina and Hafford to the North𠅊nd a background site at Waskesiu. Some acid herbicides were also investigated in South Tobacco Creek, Manitoba during 1993. Once again, maximum concentrations occurred during periods of local use (Rawn et al., 1999a). A neutral herbicide, atrazine, was also investigated in 1995 (Rawn et al., 1998). It was first detected in mid-April, peaked mid- June at about 300 pg/m 3 , and was detected until the end of October. The insecticide dacthal was identified throughout the sampling periods in 1994, 1995 and 1996 (Rawn and Muir, 1999) even though it was not used in this area (㰠 pg/m 3 ).
Non-target organisms
Pesticides are found as common contaminants in soil, air, water and on non-target organisms in our urban landscapes. Once there, they can harm plants and animals ranging from beneficial soil microorganisms and insects, non-target plants, fish, birds, and other wildlife. Chlorpyrifos, a common contaminant of urban streams (U.S. Geological Survey, 1999), is highly toxic to fish, and has caused fish, kills in waterways near treated fields or buildings (US EPA, 2000). Herbicides can also be toxic to fish. According to the EPA, studies show that trifluralin, an active ingredient in the weed-killer Snapshot, "is highly to very highly toxic to both cold and warm water fish" (U.S. EPA, 1996). In a series of different tests it was also shown to cause vertebral deformities in fish (Koyama, 1996). The weed-killers Ronstar and Roundup are also acutely toxic to fish (Folmar et al., 1979 Shafiei and Costa, 1990). The toxicity of Roundup is likely due to the high toxicity of one of the inert ingredients of the product (Folmar et al., 1979). In addition to direct acute toxicity, some herbicides may produce sublethal effects on fish that lessen their chances for survival and threaten the population as a whole. Glyphosate or glyphosate-containing products can cause sublethal effects such as erratic swimming and labored breathing, which increase the fish's chance of being eaten (Liong et al., 1988). 2,4-D herbicides caused physiological stress responses in sockeye salmon (McBride et al., 1981) and reduced the food-gathering abilities of rainbow trout (Little, 1990). Several cases of pesticide poisoning of dolphins have been reported worldwide. Because of their high trophic level in the food chain and relatively low activities of drug-metabolising enzymes, aquatic mammals such as dolphins accumulate increased concentrations of persistent organic pollutants (Tanabe et al., 1988) and are thereby vulnerable to toxic effects from contaminant exposures. Dolphins inhabiting riverine and estuarine ecosystems are particularly vulnerable to the activities of humans because of the restricted confines of their habitat, which is in close proximity to point sources of pollution. River dolphins are among the world's most seriously endangered species. Populations of river dolphins have been dwindling and face the threat of extinction the Yangtze river dolphin (Lipotes vexillifer) in China and the Indus river dolphin (Platanista minor) in Pakistan are already close to extinction (Renjun, 1990 Perrin et al., 1989 Reeves et al., 1991 Reeves and Chaudhry, 1998). In addition to habitat degradation (such as construction of dams) (Reeves and Leatherwood, 1994), boat traffic, fishing, incidental and intentional killings, and chemical pollution have been threats to the health of river dolphins (Kannan et al., 1993b, 1994, 1997 Senthilkumar et al., 1999). Earlier studies reported concentrations of heavy metals (Kannan et al., 1993), organochlorine pesticides and polychlorinated biphenyls (PCBs) (Kannan et al., 1994), and butyltin compounds (Kannan et al., 1997) in Ganges river dolphins and their prey. The continuing use of organochlorine pesticides and PCBs in India is of concern (Kannan et al., 1992 Kannan et al., 1997a Kannan et al., 1997b Tanabe et al., 1998). The Ganges river basin is densely populated and heavily polluted by fertilizers, pesticides, and industrial and domestic effluents (Mohan, 1989). In addition to fish, other marine or freshwater animals are endangered by pesticide contamination. Exposure to great concentrations of persistent, bioaccumulative, and toxic contaminants such as DDT (1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane) and PCBs has been shown to elicit adverse effects on reproductive and immunological functions in captive or wild aquatic mammals (Helle et al., 1976 Reijnders, 1986 Ross et al., 1995 Martineau et al., 1987 Kannan et al., 1993 Colborn and Smolen, 1996). Aquatic mammals inhabiting freshwater systems, such as otters and mink, have been reported to be sensitive to chemical contamination (Leonards et al., 1995 Leonards et al., 1997). 2,4-D or 2,4-D containing products have been shown to be harmful to shellfish (Cheney et al., 1997) and other aquatic species (U.S. EPA, 1989 Sanders, 1989) The weed-killer trifluralin is moderately to highly toxic to aquatic invertebrates, and highly toxic to estuarine and marine organisms like shrimp and mussels (U.S. EPA, 1996). Since herbicides are designed to kill plants, it makes sense that herbicide contamination of water could have devastating effects on aquatic plants. In one study, oxadiazon was found to severely reduce algae growth (Ambrosi et al., 1978). Algae is a staple organism in the food chain of aquatic ecosystems. Studies looking at the impacts of the herbicides atrazine and alachlor on algae and diatoms in streams showed that even at fairly low levels, the chemicals damaged cells, blocked photosynthesis, and stunted growth in varying ways (U.S. Water News Online, 2000). The herbicide oxadiazon is also toxic to bees, which are pollinators (Washington State Department of Transportation, 1993). Herbicides may hurt insects or spiders also indirectly when they destroy the foliage that these animals need for food and shelter. For example spider and carabid beetle populations declined when 2,4-D applications destroyed their natural habitat (Asteraki et al., 1992). Non-target birds may also be killed if they ingest poisoned grains set out as bait for pigeons and rodents (US EPA, 1998). Avitrol, a commonly used pigeon bait, poses a large potential for ingestion by non target grain feeding birds. It can be lethal to small seed-eating birds (Extoxnet, 1996). Brodifacoum, a common rodenticide, is highly toxic to birds. It also poses a secondary poisoning hazard to birds that may feed on poisoned rodents (US EPA, 1998). Herbicides can also be toxic to birds. Although trifluralin was considered "practically nontoxic to birds" in studies of acute toxicity, birds exposed multiple times to the herbicide experienced diminished reproductive success in the form of cracked eggs (U.S. EPA, 1996). Exposure of eggs to 2,4-D reduced successful hatching of chicken eggs (Duffard et al., 1981) and caused feminisation or sterility in pheasant chicks (Lutz et al., 1972). Herbicides can also adversely affect birds by destroying their habitat. Glyphosate treatment in clear cuts caused dramatic decreases in the populations of birds that lived there (MacKinnon et al., 1993) Effects of some organochlorines (OCs) on fish-eating water birds and marine mammals have been documented in North America and Europe (Barron et al., 1995 Cooke, 1979 Kubiak et al., 1989). Despite the continuing usage, little is known about the impacts of OCs in bird populations in developing countries. Among the countries that continue to use OCs, India has been one of the major producers and consumers in recent years. As a consequence, wild birds in India are exposed to great amounts of OC pesticides (Tanabe et al., 1998). Use of OCs in tropical countries may not only result in exposure of resident birds but also of migratory birds when they visit tropical regions in winter. The Indian sub-continent is a host to a multitude of birds from western Asia, Europe and Arctic Russia in winter(Woodcock, 1980). Hundreds of species of waterfowl, including wading birds such as plovers, terns and sandpipers, migrate each winter to India covering long distances (Grewal, 1990). While concentrations of OC pesticides in wholebody homogenates of birds have been reported elsewhere (Tanabe et al., 1998), concentrations of OCs in prey items and in eggs of Indian birds have not been reported.
A few studies related to the decline in the populations of bats in various parts of the world to OC exposure were also being conducted (Altenbach et al., 1979 Clark, 1976 Clark, 1983 Clark, 1981 Geluso et al., 1976 Jefferies, 1976 Thies and Mc Bee, 1994). The world population of bats was estimated to be 8.7 million during 1936 and it declined to approximately 200,000 in 1973 (Geluso et al., 1976) It has recovered slightly to an estimated number of 700,000 in 1991 (Geluso et al., 1976 Thies and Mc Bee, 1994). High tissue concentrations of p,p'-dichlorodiphenyldichloroethene (p,p') have been found in bats in Carlsbad Caverns in Mexico and in New Mexico in the USA (Geluso et al., 1976 Thies and Mc Bee, 1994). Occurrence of stillbirths in little brown bats exposed to high concentrations of PCBs, p,p', and/or oxychlordane was documented (Clark, 1976 Jefferies, 1976). These observations indicate that bats can accumulate high concentrations of OCs and may be affected by their potential toxic effects. The flying fox or the new world fruit bat, short-nosed fruit bat and Indian pipistrelle bat are resident species and are very common in South India. Their habitat is mainly agricultural areas, rock caves, and abandoned houses in domesticated areas. Insects constitute an important diet for many bats, allowing the passage of OCs in their body (Mc Bee et al., 1992). Several studies found OC pesticides and PCBs in livers and eggs of birds in developed countries (Becker, 1989 Bernardz et al., 1990 Cade et al., 1989 Castillo et al., 1994 Mora, 1996 Mora, 1997). Similarly, several studies reported OCs in a variety of biota including humans and wildlife from India (Senthilkumar et al., 2000). However, no study has used whole body homogenates of birds, which is important to evaluate biomagnification features and body burdens of OCs (Mc Bee et al., 1992). Earlier studies used specific body tissues to estimate biomagnification of OCs. However theoretically, estimation of biomagnification factors requires whole body concentrations rather than specific tissue concentrations.
Review of CCPs
Nearly all electronically available CCPs from each USFWS region, except Alaska, were reviewed. In total, we reviewed 226 final CCPs, 11 draft CCPs, seven Comprehensive Management Plans (CMPs) that were completed just before the 1997 mandate for CCP planning and that serve as stand-in CCPs for those Refuges, and three Conceptual Management Plans or Interim CCPs that guide the management of those new Refuges until a CCP can be completed. Hereafter, these different categories of plans are referred to generically as CCPs. A list of the CCPs reviewed is included Table S1 (Supplemental Material). To facilitate time-effective review, keyword searches of these CCPs were conducted for the terms bat, Chiroptera, Myotis, and Lasiurus. In all instances where a CCP included discussion of Myotis or Lasiurus, there was also mention of "bat," so no further searches were conducted by other generic names. Ten CCPs that did not mention bats and were unlikely to provide habitat for bats (e.g., Shell Keys National Wildlife Refuge [NWR] that consists solely of shell-derived islets with little vegetation) were excluded from further analysis. We qualitatively assessed the degree to which bat conservation and management was addressed in the CCP and assigned a score according to the criteria in Table 1.
Criteria for ranking the degree to which bat management is addressed in first-generation Comprehensive Conservation Plans (CCPs) completed as of November 2012.
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As a means of exploring correlates of planning emphasis on bats, the general linear model procedure in the statistical package R (version 184.108.40.206) was used the default settings for the general linear model were used in the analysis. The USFWS administrative region year the CCP was approved and the presence or absence of federally endangered, threatened, or candidate bat species were analyzed as covariates with CCP score. Statistical significance was assessed with an α of 0.05.
Spatial analysis
The need for strong bat emphasis in a CCP is not the same for all Refuges, and the need depends on geographically distributed characteristics that can be summarized using spatial analyses. To determine the relative importance of effective conservation planning for bats at each Refuge, we used a simple GIS analysis to generate an index of need for each Refuge. The index is based on bat species richness habitat type, as generalized by ecoregion and geological characteristics, specifically the existence of karst formations, or geologic formations shaped by the dissolution of bedrocks such as limestone or dolomite and often containing caves or sinkholes. By comparing CCP scores to this index, we were able to assess the overall adequacy of each CCP for its Refuge and to identify the units most urgently in need of improvement. Units with low CCP scores and high need indices were identified as having the most "improvement potential." In addition, we identified the Refuges most at risk from two specific threats: the spread of WNS and wind power development.
Using data sets with full coverage of the lower 48 states, we identified the following characteristics found within each Refuge: bat species occurrence according to range maps (England 2003), Environmental Protection Agency (EPA) Level III Ecoregions (EPA 2011), surface karst geology classifications (Davies et al. 1984), wind power potential according to a low-resolution wind map (NREL 2011), and the most current county-level WNS status at time of writing (Butchkoski 2012). Each Refuge was treated as the sum of its land units, such that characteristics encountered on any parcel were included in the description of the entire Refuge. Because these data are not extant for the Refuges of Guam and Hawaii, those Refuges were excluded from this analysis.
Bat species occurrence was used as the primary indicator of bat diversity and of the need for consideration of bats in CCPs. Range maps for 45 North American bat species (England 2003) were used to generate a list of species potentially occurring on each Refuge. Refuges made up of many dispersed parcels may have higher than expected bat diversity and may include unanticipated species if the Refuge includes satellite locations that are distant from the unit's core.
Ecoregion classifications provided a coarse description of both the ecological and geographic setting of each Refuge. The use of Level III Ecoregions allowed us to generalize about land characteristics such as topography, vegetation, land use, water cover, and wind power potential in a broad but simple and systematic way. These generalizations may be informative in determining the importance of particular areas to bat conservation, or the specific suite of threats likely to be encountered at a particular Refuge.
The karst classification map (Davies et al. 1984) is a full-coverage data set that we used to identify the likely location of caves or other geologic formations providing potential roosts for cave-dwelling bats. Hibernacula and other roost structures are critical to the 25 North American bat species that hibernate, and they may support winter or year-round occupancy for many species. Although karst topography does not necessarily indicate suitable habitat or present use by bats, Davies' karst assay (Davies et al. 1984) used a consistent method to identify areas likely to include caves or other potential roost structures required by cave-dwelling bats.
White nose syndrome arose as the most imminent threat facing susceptible cave-obligate bats within the zone of WNS infection after many initial CCPs were written. We use county-level WNS status classifications for each Refuge (Butchkoski 2012). This disease is likely to be a leading conservation concern for any Refuge in a county where the impacts of WNS have been documented.
To determine which CCPs are most in need of additional attention to bats, we calculated their improvement potential (IP), based on the results of the GIS analysis and their CCP scores. First, data regarding species richness (i.e., number of species present), threatened and endangered species presence, and karst topography were used to make a broad estimate of each Refuge's overall value to bats. Then, CCP scores were subtracted from this estimate to produce an IP metric. Refuges with both high value to bats and inadequate CCPs (low CCP scores) have the highest IP. Specifically, we used the following formula:
where IP is a Refuge's improvement potential metric S is number of species occurring on the Refuge E is 1 if a threatened or endangered bat species occurs on the Refuge, and 0 if not K is 1 if karst formations exist on the Refuge, and 0 if not and C is CCP score for the Refuge.
Weights were scaled to produce a range of IP metrics centered around 0, with Refuges most in need of improvement having high positive values and Refuges with the little room for improvement having negative values. The relative weighting of variables we used (e.g., 0.25 for each bat species vs. 2 for threatened or endangered bat species) was determined subjectively, with the goal of emphasizing the importance of endangered species presence and wintering habitat, while also allowing species richness to contribute substantially to IP. The resulting values are not intended to summarize the overall adequacy of each CCP (e.g., a "−6" does not indicate perfection) rather they are intended to rank Refuges and identify those Refuges with the greatest need for improvement. We did examine the use of more complex metrics, including such variables as genus richness and specific karst classifications as well as various weighting schemes with different valuations of richness, endangered species, and karst, but we found that the resulting IP rankings were effectively the same as the simple points–based formula above. For full base data used to calculate IP metric, as well as all IP scores, see Table S2 (Supplemental Material).
White nose syndrome and wind power are recently arisen threats whose future impacts are largely unknown. As such, these factors were not included in the calculation of the IP metric, but they were used to focus analysis on high-risk groups of Refuges. Separate rankings were compiled using the IP metric among Refuges with suspected or confirmed WNS, as well as those with high wind power potential. The WNS status of each Refuge was identified by converting a county-level WNS emergence map (Butchkoski 2012) into a GIS layer and overlaying Refuge boundaries. Please note that as WNS continues to spread to new areas, the data presented here will soon be outdated. Wind power potential was estimated as high, medium, or low per ecoregion, based on speed category coverage within the ecoregion, using a National Renewable Energy Laboratory 80-m onshore average wind speed map (NREL 2011). Our wind power categorizations do not necessarily identify areas most suitable for wind energy development, because wind farm siting includes a suite of additional factors not considered here, but they do identify Refuges located in regions with the potential for development based on wind resource.
Is the inhalation of ammonia a possible health problem for bats in an artificial small volume winter roost? - Biology
In North America, ''P. destructans has been found to infect at least eleven species of bats, of which it has caused diagnostic symptoms of white-nose syndrome in the endangered Indiana bat (Myotis sodalis), the endangered gray bat (Myotis grisescens), the little brown bat (Myotis lucifugus), the northern long-eared bat (Myotis septentrionalis), the big brown bat (Eptesicus fuscus), the tri-colored bat (Perimyotis subflavus), and the eastern small-footed bat (Myotis leibii''). Pseudogymnoascus destructans has been found on four additional North American bat species: the endangered Virginia big-eared bat (Corynorhinus townsendii virginianus), the cave bat (Myotis velifer), the Silver-haired bat (Lasionycteris noctivagans), and the South-eastern bat (Myotis austroriparius). The European bat species that have been shown to harbour ''P. destructans include Bechstein's bat (Myotis bechsteinii), Lesser mouse-eared bat (Myotis blythii oxygnathus), Brandt's bat (Myotis brandtii), pond bat (Myotis dasycneme), Daubenton's bat (Myotis daubentonii), Greater mouse-eared bat (Myotis myotis), whiskered bat (Myotis mystacinus), Geoffroy's bat (Myotis emarginatus), Northern bat (Eptesicus nilssonii), Lesser horseshoe bat (Rhinolophus hipposideros), Barbastell (Barbastella barbastellus), Brown long-eared bat (Plecotus auritus) and Natterer's bat (Myotis nattereri''), although large-scale European bat related fatalities are not reported.
Pseudogymnoascus destructans (formerly known as Geomyces destructans) is a psychrophilic (cold-loving) fungus that causes white-nose syndrome (WNS), a fatal disease that has devastated bat populations in parts of the United States and Canada. Unlike species of Geomyces, ''P. destructans'' forms asymmetrically curved conidia. Pseudogymnoascus destructans grows very slowly on artificial media and cannot grow at temperatures above 20 °C. It can grow around 4 °C to 20 °C, which encompasses the temperatures found in winter bat hibernacula. Phylogenic evaluation has revealed this organism should be reclassified under the family Pseudeurotiaceae, changing its name to Pseudogymnoascus destructans.
P. destructans is a psycrophilic fungus, able to grow below 10 C and with an upper limit near 20 C. This fungus produces brown and grey colonies, secretes a brownish pigment and reproduces asexually via characteristically curved conidia when cultured on Sabaouraud dextrose agar. The asymmetrically curved conidia are produced at the tips or sides singly or in short chains. Arthroconidia can be present and undergo rhexolytic separation. Research has shown that ''P. destructans grows optimally between 12.5 and 15.8 C, with an upper growth limit of about 20 C. The in vitro growth rate of P. destructans is reported to be very slow however, several studies have shown that not all P. destructans'' isolates grow at the same rate. P. destructans grows as an opportunistic pathogen on bats, causing white-nose syndrome, but it can also persist in the cave environment, as a saprotroph. P. destructans can grow and sporulate (reproduce asexually via conidiation) on keratinaceous, chitinaceous, cellulosic, and lipid/protein rich substrates including dead fish, mushroom fruit bodies and dead insects. P. destructans has been shown to utilize many nitrogen sources: nitrate, nitrite, ammonium, urea, and uric acid. Although ''P. destructans'' can penetrate senescing moss cells, cellulosic debris may not be a long term substrate for colonization. P. destructans can tolerate elevated levels of environmental inhibitory sulfur compounds (cysteine, sulfite, and sulfide), grow over a wide pH range (pH 5-11), tolerate elevated environmental levels of calcium however, ''P. destructans'' was found to be intolerant to matric-induced water stress.
Pseudogymnoascus destructans is believed to originate from Europe. The current ''P. destructans'' European distribution includes Austria, Belgium, Czech Republic, Denmark, Estonia, France, Germany, Hungary, the Netherlands, Poland, Romania, Slovakia, Switzerland, Turkey, Ukraine and the United Kingdom.
The little brown bat is also susceptible to the disease white-nose syndrome, which is caused by the fungus Pseudogymnoascus destructans. The disease affects individuals when they are hibernating, which is when their body temperatures are within the ideal growth range of ''P. destructans'',. Pseudogymnoascus destructans is the first known pathogen that kills a mammal host during its torpor. Mortality from white-nose syndrome begins to manifest 120 days after hibernation begins, and mortality peaks 180 days after bats enter hibernacula. The growth of ''P. destructans'' on bats erodes the skin of their wing and tail membranes, muzzles, and ears. White-nose syndrome causes affected bats to burn through their energy reserves twice as fast as uninfected individuals. In addition to visible fungus growth on the nose, ears, and wings, white-nose syndrome results in higher carbon dioxide levels in the blood, causing acidosis, and hyperkalemia (elevated blood potassium). Arousal from torpor becomes more frequent, and water loss increases due increased respiration rate in an attempt to remove excess carbon dioxide from the blood. The premature loss of fat reserves during hibernation results in starvation.
Severely infected bats emerge prematurely from hibernation, and if they survive long enough and enter a different hibernaculum, the likelihood of transmission is probably high, because they presumably carry a large load of fungal spores. Transmission of the infection is either physically from bat-to-bat contact, or from and hibernaculum-to-bat, through the exposure to spores of Geomyces- destructans that were present on a roosting substrate.
This species is not affected by white-nose syndrome, although the causative fungal agent, Pseudogymnoascus destructans has recently been found within their range.
Pseudogymnoascus destructans Minnis & Lindner was initially described in 2009 as Geomyces destructans by Gargas et al. In 2013, further analysis of the phylogenetic relationship moved this species to the genus Pseudogymnoascus. The conidium of this species are hyaline and characteristically curved. This species was first isolated from infected hibernating bats in New York state. Recently, this species has been isolated from cave environments no longer inhabited by hibernating bats.
Until December 2014 the cause for the abnormal behavior was unclear, as no physiological data linking altered behavior to hypothesized increased energy demands existed. The Fish and Wildlife Service published a case control study in December 2014: Of 60 little brown bats, 39 bats were randomly assigned to infection by applying conidia to skin of the dorsal surface of both wings and 21 bats remained controls. All were observed for 95 days and euthanized. 32 bats developed WNS (30 mild to moderate and 2 moderate to severe). The remaining seven infected bats were PCR-positive with normal wing histology. Infected bats with WNS had higher proportions of lean tissue mass to fat tissue mass than uninfected bats in measuring an increase in total body water volume as a percent of body mass. Infected bats used twice as much energy as healthy bats, and starved to death. Direct calculations of energy expenditure failed for most bats, because isotope concentrations were indistinguishable from background. There was also no difference in torpor durations in this experiment the average torpor duration for infected bats was 9.1 days with an average arousal of 54 min. Average torpor duration for control bats was 8.5 days with an average arousal duration of 55 min. Infected bats suffered respiratory acidosis with an almost 40% higher mean pCO₂ than healthy bats, and potassium concentration was significantly higher. Hence the following model of infection exists: Pseudogymnoascus destructans colonizes and eventually invades the wing epidermis. This causes increased energy expenditure, and an elevated blood pCO₂ and bicarbonate called chronic respiratory acidosis, possibly due to diffusion problems. Hyperkalemia (elevated blood potassium) ensues because of an acidosis-induced extracellular shift of potassium. Dying, infected cells could also leak their (intracellular) potassium into the blood. The damaged wing epidermis might stimulate increased frequencies of arousal from torpor, which removes excess CO₂ and normalizes blood pH, at the expense of hydration and fat reserves. With worsening wing damage, the effects are exacerbated by water and electrolyte loss across the wound (hypotonic dehydration), which stimulates more frequent arousals in a positive feedback loop that ultimately leads to death.
The fungus Pseudogymnoascus destructans is the primary cause of WNS. It preferably grows in the 4–15 °C range (39–59 °F) and will not grow at temperatures above 20 °C (68 °F). It is cold loving or psychrophilic. It is phylogenetically related to Geomyces spp., but with a conidial morphology distinct from characterized members of this genus. Early laboratory research placed the fungus in the genus Geomyces, but later phylogenic evaluation revealed this organism should be reclassified. The genera Geomyces and Pseudogymnoascus are closely related and found in the family Pseudeurotiaceae ''P. destructans was found to be most closely related to the Pseudogymnoascus species in 2013, implying that its name should be changed to Pseudogymnoascus destructans''.
Due to the spread of White Nose Syndrome, a fungal infection caused by the Geomyces destructans fungus in bats, including the Ozark big-eared bat, Indiana bat, and gray myotis (Arkansas' three endangered bats), as well as the brown bat and tri-colored bat, the park's caves have been temporarily closed to the public since April 16, 2010, to help slow its spread.
Bats recovering from white-nose syndrome (WNS) may be the first natural occurrence of IRIS, in a report released by the USGS. WNS is typified by a cutaneous infection of the fungus Pseudogymnoascus destructans during hibernation, when the immune system is naturally suppressed to conserve energy through the winter. This study suggests that bats undergoing an intense inflammation at the site of infection after a return to euthermia is a form of IRIS.
Formerly Cylindrocarpon destructans = Nectria radicicola
Many Pseudogymnoascus species are cellulolytic, function as saprotrophs and are either psychrophilic or psychrotolerant. Pseudogymnoascus roseus was able to form an ericoid mycorrhizal association in vitro and Pseudogymnoascus destructans infects hibernating bat and survives in the cave environment as a saprotroph. Müller indicated that all known Pseudogymnoascus species, prior to 1982, were not known to be keratinolytic.
White-nose syndrome is one of the worst wildlife diseases in recent history that is currently decimating North American cave-hibernating bat populations. This epidemic is responsible for mass mortalities in hibernating North American bats, and is caused by a uniquely cold-adapted fungus Pseudogymnoascus destructans. The fungus begins to grow on bats during the winter hibernation season, when bats are in torpor and immune-compromised. The body temperature of a hibernating bat is the optimal fungal growth environment. The fungus has affected 90% of species that hibernate in caves for the winter. Pseudogymnoascus destructans spores have been found growing on the Virginia big-eared bat. To date, no WNS mortality in this species has been observed although the syndrome has killed millions of its fellow cave-dwelling species. It is currently thought that the Big Ears may have a natural immunity to WNS through a yeast that the bat 'combs' over their body that inhibits the growth of the WNS fungus.
Some arthropods also take shelter during a fire, although the heat and smoke may actually attract some of them, to their peril. Microbial organisms in the soil vary in their heat tolerance but are more likely to be able to survive a fire the deeper they are in the soil. A low fire intensity, a quick passing of the flames and a dry soil will also help. An increase in available nutrients after the fire has passed may result in larger microbial communities than before the fire. The generally greater heat tolerance of bacteria relative to fungi makes it possible for soil microbial population diversity to change following a fire, depending on the severity of the fire, the depth of the microbes in the soil, and the presence of plant cover. Certain species of fungi, such as Cylindrocarpon destructans appear to be unaffected by combustion contaminants, which can inhibit re-population of burnt soil by other microorganisms, and therefore have a higher chance of surviving fire disturbance and then recolonizing and out-competing other fungal species afterwards.
It was circumscribed by A. Raillo in 1929 for two species, ''P. roseus and P. vinaceus''. No type specimens were retained by Raillo. In 1972, Samson designated a neotype for ''P. roseus, recognized three species (P. roseus Raillo, P. bhattii Samson and P. caucasicus Cejp & Milko) and synonymized P. vinaceus with P. roseus''. In 1982, Müller described a fourth species, ''P. alpinus''. In 2006, Rice and Currah described two additional species, ''P. appendiculatus and P. verrucosus''. In 2013, Geomyces destructans the casual agent of bat white nose syndrome was transferred to this genus and is now referred to as P. destructans. Since 2006, intensive cave sampling has identified numerous Pseudogymnoascus isolates that have yet to be described.
White-nose syndrome (WNS) caused by infection by the fungus P. destructans has increased in prevalence since 2006, mostly affecting species of bats that roost underground such as the little brown bat. The fungus, now suspected to have spread from accidental transportation by human cave workers, is thought to cause frequent arousals during bat hibernation, causing an individual to use fat stores much more quickly and die of starvation before the end of winter. WNS can affect ''T. brasiliensis'', but has yet to be greatly introduced to their habitat due to their preference for more arid caves. WNS has low prevalence in the subtropic and tropic regions where ''T. brasiliensis'' resides.
Several species of fungi infect the roots of waratahs, causing significant plant morbidity or death. Typical symptoms include yellow leaves, wilting, blackening and dieback or part or all of the plant, or lack of proteoid roots. The most common pathogen is the soil-borne water mold Phytophthora cinnamomi, which appears to be more problematic in cultivated plants than in wild populations. Mass plantings at the Royal Botanic Gardens in Sydney and at Mount Annan planted before the 2000 Summer Olympics were devastated by the disease. Rhizoctonia solani can cause damping off or root rot, and is an uncommon pathogen. Cylindrocarpon scoparium and ''C. destructans (now Nectria radicicola'') are also uncommon causes of infection and result in decay of the crown of the plant. Although significant problems, fungi are less likely to be the cause of plant morbidity than poor drainage or soil conditions.
A positive breakthrough may have come while utilizing competitive genetics to investigate the evolutionary history of ''P. destructans'' compared to six closely related nonpathogenic species. The study published in the journal Nature in 2018 discovered that due to a lost enzyme, ''P. destructans'' lacks an ability to repair DNA which has been damaged by ultraviolet (UV) light. Ongoing research is taking place to see if there is a practical method to have bats activate a UV system as they enter and leave a hibernaculum and treat their infection. Not a long term solution, it may be enough to avoid population collapse allowing the species to evolve its own defenses to the fungus as Eurasian bats have.