**4. Chiropteran physiology**

#### **4.1 Skeletal morphology and flight**

Unique skeletal system and integumentary system adaptations combined make bats the only mammals capable of true, sustained flight. Chiropteran species have a strong axial skeleton with non-pneumatized, marrow-filled long bones and elongated metacarpal bones and phalanges [64]. A bilayered 1–2 cell thick epidermis (e.g. <s10 μm in the epauletted fruit bat, *Epomophorus wahlbergi*) separated by a non-distinct dermis or hypodermis form the extremely thin wing membrane. Within the non-distinct dermis there are highly vascularized connective tissues [65]. Collagen and elastic fibers within the connective tissues serve as "fingerprints" that can be used to identify individuals [66] and are responsible for providing the mechanical strength needed for flight [65, 66].

The wings may serve additional purposes other than flight. The thin, and highly vascularized wing tissue has been suggested to allow diffusion of oxygen and carbon dioxide between the environment and the vasculature, thereby contributing to a bat's total gas exchange [65]. Wing membranes are essential to thermoregulation [67] and also water exchange [8, 65]. These adaptations allowing water and gas exchange through the wing membrane could also increase the risk of dermal absorption of pesticides—especially considering the wing membrane of bats accounts for up to 85% of a bat's total body surface [65].

When bats fly, it is energetically expensive, especially since bats flap continuously rather than glide [36]. To meet this energy demand, food intake is high. When foraging each night, frugivores consume up to 2.5 times their body weight in fruit [68], insectivores consume up to 1.3 times their body weight in insects [7], and some nectivorous species consume nectar from 80 to 100 flowers each night [69]. With high food consumption, there is an increased risk of oral exposure to all bat species.

#### **4.2 Torpor**

Torpor or heterothermic responses are "facultative, reversible decreases in metabolic rate and body temperature in response to environmental cues" [70]. All species of bats may select to employ daily torpor, the lowering of their body temperature set point, respiratory rate, and metabolic rate for a single circadian cycle or a portion of a circadian cycle [49, 71] to help offset the high metabolic and thermoregulatory costs [71]. Hibernating species may engage in multi-day torpor bouts which can save them up to 99% of their daily energy requirements [72]—allowing them to survive winter when food availability is reduced [71].

Throughout hibernation (multi-day torpor), bats use white adipose tissue as their primary fuel source [73]. When arousing from torpor, especially multi-day torpor bouts, they may engage in passive rewarming which can reduce arousal costs by 50% or more [74]. Even with passive rewarming, torpor arousal is still energetically demanding and bats must mobilize a portion of their brown adipose tissue to generate heat [73].

During the fall, when hibernating bats are developing large fat stores, the risk of oral exposure to pesticides increases as they consume a greater amount of prey. Concurrently, lipophilic (fat soluble) pesticide residues are stored within the adipose tissue (see Table 1 in [30] for studies which measured pesticides in Chiropteran adipose tissue) as a large portion of pesticides, especially within the insecticide class, are lipophilic [75]. Hibernating bats may be at risk of continuous lipophilic pesticide exposure as bats mobilize their white adipose tissue. During arousal the rapid mobilization of brown adipose tissue may cause an increased risk of a sudden large dose of lipophilic pesticides to be released.

*Pesticide Exposure Risks to Chiropteran Species and the Impacts on Emerging Zoonotic Diseases DOI: http://dx.doi.org/10.5772/intechopen.100643*

## **4.3 Echolocation**

It is a common myth that bats are blind, as in fact they have color vision and may be sensitive to UV light [76]. The majority of species within the Pteropodidae family (Old World Fruit bats such as flying foxes), use their acute vision to perceive their environment and forage for food rather than rely upon echolocation. Whereas bats in the other families are believed to use echolocation as the primary way to forage and navigate through their environments [77]. When these species echolocate, they produce high frequency sounds or tongue clicks [78]. Their auditory system processes the echoes and enables the 3D perception of the object and the spatial location of the object. For more detailed information on the adaptive sonar behavior of bats and echolocation auditory mechanisms see [77, 79].

Echolocation has been suggested to contribute to the global success of a wide range of bat species [78]. While echolocation may not increase a bats risk to pesticide exposure, it can be negatively affected by sublethal concentrations of pesticides [80].

#### **4.4 Other considerations for reference species**

At sublethal doses, the neonicotinoid imidacloprid suppresses the expression of genes related to echolocation (e.g. FOXP2) [80], interferes with the spatial memory [81], and leads to inflammation and mitochondria dysfunction-related apoptosis [80]. This type of ecotoxicological data suggests that using mammals who do not have echolocation capabilities may need to be reassessed as a reference species for bats.

Other considerations for reference species selection should consider that other mammals do not have the physiological adaptations described above. Additionally, rats, the most commonly used reference species, have a relatively short lifespan of 3 years or less in a laboratory or pet setting, with the average lifespan of a wild rat being less than 1 year [82]. Free-ranging bats live much longer with some species living for at least 32 years based upon recapturing banded individuals [83]. The longer lifespans of bats put them at increased risk of bioaccumulation of pesticides and other contaminants such as heavy metals.

## **5. Zoonotic disease risks**

It is estimated that over 60% of newly emerging diseases are zoonotic with approximately 3 out of every 4 diseases being the result of spillover from wildlife [84, 85]. Due to the ongoing worldwide pandemic COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), bats are often cited by the media and some scientists as the source of this zoonotic disease as well as other zoonotic viral diseases such as Ebola [86]. COVID-19 is an emerging infectious disease but at the time of this writing there is no evidence it is a zoonotic disease [87].

A zoonotic disease, or zoonosis, is defined by the World Health Organization as "any disease or infection that is naturally transmissible from vertebrate animals to humans [88]". While the origin of SARS-CoV-2 remains unknown [89], there is no evidence that bats serve as the reservoir of the virus. This is because a reservoir is "a population, species or community assemblage of different species in a given geographic area in which a microorganism naturally occurs and is indefinitely maintained" [86]. Since SARS-CoV-2 has not been detected in any bat species, they cannot serve as reservoirs of the virus [86]. The miscommunication seems to originate from the misunderstanding of a report of a newly identified coronavirus circulating within intermediate horseshoe bats (*Rhinolophus affinis*) which exhibits 96% similarity to SARS-CoV-2 virus [90]*.* This newly identified coronavirus is suggested to be the ancestral origin of the SARS-CoV-2 virus [90], but it is not the same virus that is causing the COVID-19 pandemic. For a complete review on effective communication regarding zoonotic diseases and the terminology commonly misunderstood and miscommunicated in relation to bats see [86].

While COVID-19 may not be a zoonotic virus, bats do serve as the reservoir for other zoonotic viruses such as rabies and Nipah virus [86]. When new zoonotic diseases arise, they are most often the result of a spillover event from bats or other wildlife reservoir species to humans or another mammalian host [10, 91].

Spillover events are complex processes and are rare [9]. When spillover events do occur, there must be a "perfect storm" or a situation where several factors need to be present for the spillover event to occur. Two of those factors, pathogen shedding and environmental conditions, are discussed below. For an extensively detailed description of all potential factors see Plowright et al. 2015 [92].

Before describing these factors, it must be noted that the immune system of bats is not well understood. The limited research conducted on Chiropteran immune systems verify there is much diversity among species (e.g. the interferon gene locus is contracted in the black flying fox (*Pteropus alecto*) whereas expanded in the Egyptian fruit bat (*Rousettus aegyptiacus*) [93–95]). While this diversity exists, their immune systems uniformly appear to allow chronic/latent viral infections where the host remains asymptomatic and viral shedding does not occur. This benign virus-host relationship is more commonly recognized as the Susceptible-Infectious-Latent-Infectious (SILI) hypothesis [10].

The SILI hypothesis suggests that when bats are stressed by physiological and/ or environmental stressors, the virus(es) can reactivate due to a suppression of the host's immune system [10]. These viruses may remain nonpathogenic within bats, but the reactivation of the viruses results in viral shedding [10]. Viral shedding leads to an increased risk of spillover events as these viruses may cause severe disease in other mammals [86, 96].

Anthropogenic changes such as urbanization and high human population density are known physiological and environmental stressors to bats due to the loss, degradation and/or fragmentation of suitable habitats [97]. While these anthropogenic stressors are well studied, there are few experimental studies assessing if pesticides and other pollutants are sublethal stressors as most Chiropteran contaminant studies are observational.

Pesticides are classified as stressors in other mammalian species as they are known to cause oxidative stress, genotoxic effects, and suppress the immune system [98–101]. Two studies conducted in the fruit bat *A. lituratus* measured oxidative stress when exposed to fruit sprayed with the organochlorine insecticide endosulfan and the pyrethroid insecticide deltamethrin. The metabolism of the pesticides increased the generation of reactive oxygen species (ROS) including nitric oxide and hydrogen peroxide leading to liver morphological changes [102] and oxidative damage to both liver and the pectoral muscle [102, 103]. Since fruit bats are believed to be highly resistant to oxidative stress in part due to the high level of antioxidants in their diet [104], this finding is significant. Especially since it is well established that chronically increased oxidative stress leads to a variety of pathophysiological conditions [105] and directly alters the immune system [106]. It is unknown if increased oxidative stress leads to increased viral shedding, thereby increasing the risk of zoonotic disease and should be evaluated in future studies.

Oxidative damage has been shown to induce genotoxicity when humans have been exposed to some contaminants such as lead and toluene [107, 108]. Two commonly employed genotoxicity tests are the micronucleus test and the comet assay. The micronucleus test assesses chromosomal damage (see [109] for detailed review

#### *Pesticide Exposure Risks to Chiropteran Species and the Impacts on Emerging Zoonotic Diseases DOI: http://dx.doi.org/10.5772/intechopen.100643*

of the technique) while the comet assay detects a wide array of DNA damage such as DNA strand breaks (single and double stranded) and incomplete excision repair (see [110] for detailed review of the technique).

Micronucleus tests conducted in the insectivorous bat species, Parnell's mustached bat (*Pteronotus mexicanus*), documented that *P. mexicanus* roosting in caves surrounded by agriculture and foraging over pesticide treated fields, had a higher number of micronuclei than bats roosting and foraging in areas where no more than 15% of the surrounding land-use was agriculture [111]. Another study assessing micronuclei and other nuclear abnormalities in insectivorous, frugivorous, and nectivorous bats discovered that nectivorous bats did not have any significant nuclear changes in agricultural or urban areas [112]. Frugivorous and insectivorous bats however did have significantly higher micronuclei in urban and agricultural areas with other nuclear changes such as binucleated cells being commonly observed in urban areas [112]. A third study assessing genotoxicity using the comet assay documented that banana bats, *Neoromicia nana*, foraging over South African waste water treatment plants containing pesticides and other pollutants had significantly higher DNA damage compared to bats foraging at reference sites within Umdoni Park [113]. All three studies are limited from the perspective that none measured actual pesticide concentrations in tissues or bodily fluids (e.g. urine). Although it cannot be solely concluded that the genotoxic effects were truly from pesticide exposure, research in other mammalian species documents a clear link between pesticide exposure and induced genotoxicity [99]. This fact identifies along with the three Chiropteran studies suggesting pesticide exposure can induce genotoxic changes, highlights a large research need for assessing if pesticides are genotoxic to bats and if it leads to viral shedding via immunosuppression mechanisms.

Although there are few studies measuring the ecotoxicological effects of pesticides on bat species, the available studies show that some species suffer oxidative damage, genotoxic changes, and mortality. This suggests that compounding anthropogenic stressors, including environments with heavy pesticide use, may cause environmental conditions to become favorable for a spill-over event to occur by causing physiological stress in insectivorous and frugivorous bats. This compounded physiological stress may result in viral reactivation and viral shedding by suppressing bats' unique immune system. Because bats sustain a high viral load and are phylogenetically close to humans, the risk of zoonotic disease transmission from spill-over events is greatest from bats compared to other species [114].
