Italian crested newt

Italian crested newt
Juvenile with yellow dorsal stripe
Conservation status
Least Concern  (IUCN 3.1)[1]
Scientific classification
Kingdom:	Animalia
Phylum:	Chordata
Class:	Amphibia
Order:	Caudata
Family:	Salamandridae
Genus:	Triturus
Species:	T. carnifex
Binomial name
Triturus carnifex (Laurenti, 1768)
Range of T. carnifex in Europe

The Italian crested newt (Triturus carnifex) is a species of newt in the family Salamandridae.

Habitat

Triturus carnifex is found in parts of the Balkans and Italy. It is an aquatic breeder that can spend up to four months in the water.^[2] The location of the ponds where they breed affects the time when T. carnifex enters the water.^[2] T. carnifex prefers living in deep water since it is a nektonic species: it swims freely and is independent of currents.^[3] The absence of predatory fish may also explain why T. carnifex is inclined to ponds, rather than other larger bodies of water.^[3]

They typically prefer ponds in northern Europe, where temperatures are colder. Adult T. carnifex start to arrive between February and May, and leave between July and October.^[4] In warmer ponds, adult newts arrive within a month and leave during a two-week period in July.^[4] Andreone and Giacoma (1989) speculated that newt migration into the ponds increases after rainy days, since after rainfall, newt activity is not limited by humidity.

Higher altitudes, where temperatures begin to decrease, have a direct effect on the size of T. carnifex.^[5] Ficetola et al. (2010) discovered that living in colder temperatures resulted a body size increase in both male and female. Females in higher altitudes were found to be larger because they were carrying more oocytes and larger ovaries, which gave them a reproductive advantage over the smaller females.^[5] T. carnifex is poikilothermic and larger body sizes help to reduce heat fluctuations.^[5] Ficetola also found that fluctuations in body temperature of T. carnifex decreased when body size increased. An increase in body size also occurs where there is increased precipitation or nearby primary producers, due to the effect of increased resources on animals.^[5]

Human interference has dramatically changed the habitat of T. carnifex due to the expansion of industrial areas and urban centers.^[3] This results in a fragmentation of natural landscapes, which leads to selective extinction, genetic drift, and inbreeding from isolation.^[3] Introducing fish to isolated wetland habitats also leads to a decline of amphibians because of predation on newt larvae.^[3]

Respiration

T. carnifex is a pond-dwelling species of salamander that has adapted to living in temporary or polluted ponds.^[6] As a result, adult newts in the genus Triturus have multiple ways to respire. The three methods of oxygen intake are via the skin (the primary source), lungs, and buccal cavity.^[7] This is due to their poorly vasculated, elastic, simple lung structures, although the lungs are more vasculated at the posterior end.^[7] This is important when there are poor oxygen conditions, as it causes the newts to use pulmonary respiration as their primary source.^[7] In addition, newts also use pulmonary respiration in active metabolic states such as courtship, breeding, or food gathering, when they ventilate at 3-minute intervals.^[7]

When newts are inactive or in oxygen-rich environments, respiration can be done through their skin^[7] which accounts for 74% of the cutaneous respiration.^[8] However, oxygen-poor environments, such as stagnant ponds, pose a threat to the survival of larval newts.^[9] This is because oxygen molecules do not readily dissolve in water, and as T. carnifex is an immobile animal, extracting oxygen from water is difficult.^[9] Newts consequently have external gills at birth that persist until metamorphosis, which assists survival in low-oxygen conditions.^[6]

The gills have their origins in gill arches 2, 3, and 5; these increase with 3 more bars, that consist of secondary filaments, which create the respiratory organ.^[6] In the secondary lamellae there are connective tissues that provide support to the epithelium; these make up the water-blood barrier, in particular where the capillaries are immediately below the basal cells.^[6] There are numerous secondary filaments along with the main filaments, which are lengthened to extend the oxygen-catching capability.^[6] This increase in surface area counteracts the decreased oxygen available, by allowing greater oxygen uptake.^[6] In larval stages, the gills are well-developed with a great number of respiratory lamellae.^[6] This adaptation is hypothesized to be an advantage in response to the conditions in an aquatic environment.^[6]

In the gills, there are neuroepithelial cells, which are important for sensing oxygen during developmental and adult stages.^[6] The gills also contain Leydig cells.^[6] Though the function of these cells remains unclear, the high abundance of them in pond-dwelling species, which live in a low oxygenated environment, in comparison to stream-dwelling species, which live in a higher oxygenated environment, can lead to the conclusion that Leydig cells form in response to these conditions.^[6] Different hypothesis have been formed with regards to these cells. One suggests that they retain water, another saying that it secretes mucus.^[6] However, it uses mostly cutaneous respiration as an adult, which enables it to thrive.

There are still other adaptations that enable the newts to survive.^[6] T. carnifex have made physiological adaptations that enable them to stay in hypoxic conditions or meet their increased metabolic needs.^[10] This compensation is in the larvae as soon as they are born.^[10] Spleen size can increase as the temperature declines for adults – in larvae, there is no dramatic change in spleen size.^[10] This difference is attributed to the improbability of a larvae newt ending up in a hypoxic environment primarily due to gill movement.^[10] The spleen plays a large role in this adaptation; 50% of the blood is redirected into the spleen when the conditions in their habitat are well oxygenated.^[10] When the large stores of erythrocytes dips below necessary, the spleen of T. carnifex will release red blood cells into the blood stream, which is caused by hypoxia or increased metabolic needs.^[11] This very specific compensatory respiration – the ability to adapt to respiratory conditions – is known as hypoxia, and increases the red blood cells as a method of maintaining oxygen levels in an oxygen-lacking environment.^[11] These adaptations have evolved due to both the environment and the physiological conditions the newt finds itself in.^[11] This can be seen when the animal releases red blood cells into the bloodstream as a method of maintaining oxygen levels.^[11] This feature is commonly shared among amphibians, and it is possible that all ectotherms are capable of this.^[11]

In the category of ectotherms, T. carnifex is a heterothermic animal, meaning its internal temperature varies with differing climatic temperatures. This mechanism causes the newt to adapt to the changing temperatures by the increase of liver pigmentation in the winter months.^[12] Liver pigment cells play an important role in scavenging ions and free radicals in the newt, including the uptake of oxygen.^[12] During the winter months, the newt enters a state of hibernation, its respiratory rate slowing, the amount of oxygen intake decreasing.^[12] The increase of these liver pigment cells allow for storage of oxygen, as well as other important ions and free radicals.^[12]

Chemical effects on the respiration of Triturus carnifex

As an aquatic species of newt, T. carnifex requires areas of standing water such as ponds, lakes, farmlands, etc., for its habitat. It is very susceptible to slight changes in its environment, and is thus considered an ideal bio-indicator species.^[13] The species is thought to be on the decline due to an increase in industrialization and housing development, both of which interfere with the habitat of the newt.^[14] The greatest threats to the newt are loss of aquatic habitats (most importantly, breeding sites), agricultural pollution and intensification, and the inclusion of new predatory fish into its habitat. The intrusion of humans not only physically destroy their habitats, but introduces chemicals and pollutants that have lethal effects on amphibian populations at what is normally considered a safe amount.^[13] Due to its wide distribution, however, extinction of the species is considered to be of small concern at present, even though the populations have been decreasing.^[14]

The larvae of the newt are entirely aquatic and have non-directional external gills, with a great number of lamellae throughout the main gill filament.^[6] This makes the larvae even more susceptible to pollutants, poisons, and changes in its habitat than adult T. carnifex as larvae must remain in the water until their lungs have developed and their external gills have disappeared. In this way, the larval newt is exposed to any contaminants present in the water from the moment of its birth.

The heavy metal cadmium has been shown to be especially detrimental to T. carnifex even at a level of concentration that falls within the freshwater safety values of 5 µg/L established by the Italian Ministry of Health and the European Community.^[13] Chronic exposure to these levels of cadmium was found to significantly decrease adrenal gland activity in T. carnifex, resulting in an endocrine-disrupting effect and affecting hormone production, which is suggested as a possible explanation for the decline of the species.^[13] Cadmium is known to be present in the environment due to industrial and consumer waste entering into aquatic and terrestrial systems, and has a very long half-life of around thirty years. It is also genotoxic, meaning it builds up in the kidneys and liver, thus accumulating in the food chain. It can damage physiological processes or tissues in aquatic organisms even at concentrations significantly below the lethal level, and is embryotoxic – that is, it causes various malformations in embryos and lethality in all forms of animals.^[13]

The amphibian adrenal gland is responsible for the release of corticosteroids and catecholamines which aid in stress response in the newts by maintaining ion and water balance. This allows them to adapt and moderate environmental changes in their habitats. However, the introduction of cadmium into aquatic systems has been shown to disrupt normal operation of the adrenal gland in amphibians and result in a marked drop in ACTH (a hormone secreted by the anterior pituitary gland which triggers an increase in production of corticosterone) and therefore also a drop in corticosterone production. A drop in these necessary hormones severely affects the survivability of the newt, and its hypersensitivity towards chemical and pollutant exposure serves to cripple T. carnifex's longevity further.^[13]

Physical effects on the respiration of Triturus carnifex

Temperature can affect T. carnifex's respiration by an interaction with the cardiovascular compartment of the newt. This is mainly seen by the metabolic changes that occur with altering temperatures. When there is an increase in temperature, metabolic rate will increase, as will the heart rate. This occurrence will cause the need for higher intake of oxygen. The system must react accordingly, whether it is through cutaneous, lung, or ventilator means will depend on other physical factors it is dealing with.

One of these “other factors” is the presence of oxygen in the environment in which T. carnifex resides at the time of analysis. In an oxygen-low environment, the organism will need to undergo splenic activity in order to remain oxygenated. When the newts enter a hypoxic environment, their spleen will decongest to allow erythrocytes out and into blood stream. This process will increase hemoglobin in the blood, and therefore the oxygen-carrying capacity. Once this has occurred, T. carnifex are able to survive in oxygen poor environments. Upon re-introduction to a normoxic environment in which there is satisfactory oxygenation, the spleen will regain the red blood cells, reducing the hematocrit.^[11] This process exists only in adulthood.

Development brings many changes about in the Italian crested newt. One of these is the alteration from oxyconformer to oxyregulator. Obviously, this difference will change how the newt deals with changes in its respiratory conditions. Another developmental change is the loss of external gills from juvenile stage to adulthood. This affects T. carnifex as the difference between being able to outreach the DBL (juvenile) and the inability to do this (adult). This inability introduces a diathesis to hypoxia in stagnant water. This is because there is a lack of convection in the water, and the oxygen is being subtracted more quickly than it can diffuse (due to oxygen’s slow diffusion constant through water).

Circulation

Basics of the circulatory system

The heart of Triturus carnifex, like that of most amphibians, consists of two atria and one ventricle.^[15] Blood flows from the anterior and posterior caval veins into the right atrium; blood that entered the heart from the left atrium is then expelled out of the ventricle.^[15] Newts do not have a coronary artery on the ventricle, due to circulation that is found in the conus arteriosus.^[15] Newts contain a special circulatory adaptation that allows them to survive ventricular penetration.^[15] Myachi found that when a newt’s ventricle is punctured, the heart will divert the blood directly into an ascending aorta via a duct located between the ventricle and the conus arteriosus. T.carnifex begins to regenerate the ventricle by a thickening of the epicardial layer that protrudes to allow the new vessels to form, and concludes with a regeneration of the entire myocardial wall.^[15]

Blood flow physics are determined by certain formulas which can be used to find specific values. LaPlace’s Law, Pouseuille’s Law, and the Law of Bulk Flow all are necessary to find values of the cardiovascular system. Blood volume is a factor necessary for function and survival of an organism. This volume is regulated through negative feedback mechanisms. The circulatory system has many functions, such as bringing necessary substances throughout the body and distributing them where needed. Another function is the providing of hydraulic pressure to certain organ systems in the organism.^[16]

Circulatory mechanisms in altering physical conditions

T. carnifex is a member of a large family who all share common traits, one of which is the ability of this organism’s spleen to store large volumes of erythrocyte when the oxygen available goes beyond what is needed. Conversely, when the oxygen supply dips below necessary, the spleen of T. carnifex will release the red blood cells into the blood stream (as in cases of hypoxia, see below).^[17] This process will affect many aspects of the system, including the composition and volume of the blood. The surroundings of the animal will affect it. These surroundings include both the environment, and the physiological conditions the newt finds itself in. One such instance of this regulation is known as compensatory respiration, found during hypoxia. Hypoxia happens when there is an extremely low or limited amount of oxygen available to an organism.^[17]

During compensatory phenomena, many factors of an animal may become subject to change. One of these is the blood volume, which can vary anywhere from 6-8.5ml/100g of body weight.^[11] The blood volume of T. carnifex has a direct relationship with the blood pressure of the animal, and so when there is a change in blood volume, there will also be a change in blood pressure. Because the vascular structures and systems (referred to as the vascular components) have dilatability, changes in blood pressure are permitted to allow for changes in blood volume.^[17] One hypothesis is that blood pressure is vital and defining in the storing or releasing of oxygen to and from the spleen.^[11] This is possible, because no structure (anatomical, histological, or otherwise) has been found that could explain this compensatory phenomenon, and because blood pressure would have no such structures, it becomes a strong component for the responsibility of the splenic activity. In T. carnifex, respiration in the lungs is not nearly as important as the cutaneous compartments.^[17] Much research has taken place with anesthetized T. carnifex, and under these circumstances cutaneous respiration is the sole method of breathing.

In periods of stress, catecholamines (adrenaline, noradrenaline, and dopamine) are released into the circulatory system in an attempt to minimize the stress on the physiological systems.^[18] Multiple environmental factors influences the amount of noradrenaline and adrenaline that is secreted such as dehydration and hemorrhage that cause an increase in these substances.^[18] In T. carnifex, circulatory adrenaline and noradrenaline increase when forced to participate in activities, and the presence of fungicides into the circulatory system decreases catecholamine content.^[18] Conversely, acetylcholine elicits noradrenaline or adrenaline secretion in T. carnifex.^[18] In amphibians the increase in catecholamines can cause high blood sugar because of stimulation of the liver and muscles.^[18]

A minimum of 4000 heartbeats of the newt is the necessary amount for haematological parameters to become stable when there is a shift in respiratory conditions. The colder the temperature, the harder it seems for T. carnifex to reach this 4000 beat minimum. In an experiment performed by Frangioni et al. it was observed that at 6 degrees Celsius, it took 360 minutes, whereas at 18 degrees Celsius, it only took 150 minutes.^[11]

When T. carnifex are in an optimal respiratory environment (that is, exposed to air), they contain low RBC counts, as well as low hematocrit values. This implies that in these conditions, the spleen is very congested. As stated, this is because in areas of high oxygen, the newt is not in hypoxia and does need to compensate for the lack of oxygen in its surroundings. Therefore, release of oxygen from the spleen is unnecessary and not triggered. The opposite is seen when the animal is placed in a hypoxic environment. There is a release of erythrocytes from the spleen and the values in the blood are raised to high.^[17]

Every time there is an increase in blood volume (created by the release of erythrocytes), there is an increase in blood pressure, and therefore they are said to have a direct relationship.^[17] Thus, if one increases, so will the other. Besides blood volume, a factor affecting blood pressure is the temperature. At lower temperatures, the newts will have a lower blood pressure, and the opposite is true for a higher temperature. Another factor is the congestion of the spleen. When congestion of the spleen rises, the pressure will fall; and vice versa.^[17]

During hypoxia, there is a great deal of arterial pressure which appears when there is a quick change from an oxygenated environment to a hypoxic one. That increase is due to the swift decongestion of the spleen as it releases erythrocytes into the bloodstream to allow respiration within the newt.^[17] However, this increase in pressure occurred only at the 6 degree and 12 degree mark; at 18 degrees the blood actually decreased.^[11] This suggests that even though blood pressure is key in determining respiratory compensation, it is by no means the only factor, or even the most important. Although there is a decrease in blood at 18 degrees, the spleen is still emptied (as it is at any temperature). There is a decrease in blood plasma when this occurs so as to keep blood volume at a constant, or even a lowered value.^[17]

In well oxygenated environments, temperature can be seen working alone, without the added challenge of a hypoxic environment. When an increase in temperature occurs in the surrounding environment of a newt, this increases metabolic activity. When there is such an increase, the spleen will empty itself of erythrocytes and the volume of red blood cells will go up. This is to assist with the greater need for consumption of oxygen.^[17] In a high temperature environment where T. carnifex is exposed to air, it can be expected that there will be a large increase in blood volume. However, the blood plasma will decrease and prevent an increase in blood pressure.

In terms of compartmentalization of the newt’s respiratory systems, the systemic (lungs) and cutaneous (skin) systems work and function independently. Despite this, there is a very high correlation between the two with respect to their abilities in times of hypoxia. Both systems attempt to obtain as much oxygen as possible – cutaneous through vasodilation, and systemic through splenic decongestion.^[17]

Cardiovascular system response to hibernation

During the winter period, the physical environment of T. carnifex will change drastically, and a need for adaptation to this will arise. The replicatory processes of cells are lowered a great deal during hibernation, likely due to the decrease in metabolic activity and therefore a low need for replication.^[19] Cells will exhibit features associated with a process known as “aging”. This involves dropping elements that are less mature from the circulatory compartment. Aging is assisted by apoptosis (programmed cell death, “cell suicide”). As stated, metabolism is severely lowered during this winter period. This decrease is demonstrated by noticeable changes both in blood circulating cells and haemopoietic cells (these are cells involved in all blood cell production, stem cells found in bone marrow).^[19] There are other decreases seen in these cells, and these include enzymatic activity. Because it is necessary for some supplementation of this, the liver increases in activity during this period. It is as if there is a shift in responsibility from the haemopoietic cells to the liver. There is a high correlation between increased activity of the liver and decreased myelopoietic activity.^[19] Decrease in metabolic activity could be due to the low motility or movement of newts during winter. Cold temperatures seem to slow down their system.

Cardiac regenerative capabilities

The most basic aspect iof the heart s the cardiovascular compartment’s ability to facilitate and maintain circulation. The structures of the heart play a vital role in its ability to do this. Important structures include:the muscular wall that has the function to contract and therefore pump blood throughout the organism (this wall is both vascularised and innervated, and n newts, innervated mainly by the vagus nerve,)^[20] the inside endothelial layer called the endocardium, and the outside mesothelial layer called the epicardium.^[21] Contraction of the heart is due to the electrical stimuli (signals) that are received from the central nervous system in organisms.^[21]

More specific to amphibian hearts, the capacity to regenerate is actually higher than many animals, humans included. This ability allows organisms to survive trauma to the heart. In newt hearts, this ability is not as impressive. When there is injury to the heart muscle, scar tissue will form and weaken the heart, making it more susceptible to later problems. However, some regeneration does occur.^[21] In later testing, it was discovered that when the trauma is to the base region of the heart, regenerative capacities are of a much stronger nature. The evidence of regeneration is seen in the production of DNA. This shows that new cells are being made to reconstruct the tissue that was lost or damaged during injury to the cardiac muscle. Furthermore, the presence of mitotic figures seen defines division and creation of new cells. These appear in the striated muscle of the heart.^[21]

The bottom line of these phenomena is that newts are capable of cardiac regeneration, but it is very dependent on where the damage occurs, because of the differing capability of different regions of the heart in the creation of new cells and building of new tissue. In short, where the damage occurs is directly related to how much regeneration will occur.^[21]

Circulatory response to feeding

There are broad generalizations relating to amphibians that can be used to describe their digestive processes. One such generalization is that digestion raises metabolic rate in the organism in which it is occurring. This wide idea will apply to T. carnifex. Digestion results in a hypertrophy (swelling) of the gastrointestinal regions.^[20] This is similar to the shunting of blood flow to the gastrointestinal compartments, as seen in muscular exercise. Both activities place a specific demand on the cardio-respiratory system. However, the similarities end at the surface and underlying mechanisms between the two processes differ. While muscular activity increases heart rate by vagal release, the digestive processes act directly on the heart and involve both non-adrenergenic and non-cholinergic factors.^[20] Digestion is demonstrated by an increase of the concentration of HCO3 in the blood. Since this shift is towards the lower (and more acidic) end of the pH scale, there is a countering mechanism in the increase of CO2 pressure in order to reduce the pH shift in the arterioles. This another example of regulation as opposed to conformation in the Italian crested newt.^[20]

The high metabolic activity is caused by the digestion after feeding and more specifically the absorption of nutrients into the body and the utilisation of those nutrients. The height of metabolic change is directly proportional to how much food is consumed by the newt. There will also be an increase in heart rate post-feeding.^[20]

An important note regarding the heart of the newt is that it possesses an incomplete separation between the two sides. This is not ideal, as it makes it possible for oxygen rich blood and oxygen poor blood to mix. The mixing of these two differing compositions would create problems in the arterial blood/gas composition, such as the decrease and reduction of the metabolic rate which is sustainable at the maximum level.^[20] Because of this, the physiology of newts attempts to avoid mixing.

Circulatory alterations in various physical conditions

Requirements of the circulatory system can alter with the different environments T. carnifex finds itself in. This is because in separate conditions, different things are required to assist with functioning of T. carnifex, specifically with the functioning of the cardiovascular system. The requirements of the blood compartment will change when the environment of the animal tampers with the blood’s oxygen-carrying capabilities.^[22]

The movement of oxygen molecules into the tissues of the newt from the surrounding medium occurs in two distinct stages:

1. The medium containing the oxygen will undergo convection.
2. The internal medium will undergo convection.^[22]

Development of the cardiovascular system

In early stages of development in amphibians, it has been discovered that convectional gas transport, the method through which hemoglobin is carried throughout the body, is an unnecessary process. This is important because it indicates that the coupling of ventilator gas transport and hemoglobin gas transport are independent mechanisms and not yet coupled as they are in adulthood.^[23] An example of this difference is seen in the response to hypoxia between organisms at differing developmental stages. In juvenile newts, there is no cardiovascular response in conditions of hypoxia.^[23]

T. carnifex displays a unique hemoglobin and red blood cell (RBC) adaptation in their circulatory system.^[24] When newts are induced into anemia, they are able to respire without the need of blood cells, or their body making blood cells.^[24] Instead, around two weeks after anemia is induced T. carnifex produces a mass of cells that helps to revitalize the already circulating red blood cell mass.^[24] Casale et al. found that red blood cells of the newt not only produce hemoglobin, but also ferritin, ribosomal proteins, and proteins assumed to be catalase. These extra proteins were produced in high concentrations in early RBC, but decrease quickly as the cell matures.^[24] Casale et al. also found that although hemoglobin production decreases as the RBC matures, the cell creates more protein to aid in hemoglobin production.

T. carnifex is likely to develop in areas of varying temperatures and changing oxygen availability. This is an important piece of knowledge about the typical environment of T. carnifex: from a very early age, the species is exposed to situations in which they will need to be able to regulate their oxygen levels.^[23]

Continuing with developmental changes, T. carnifex will undergo a coupling of many processes as it eases into adulthood. Some of these processes include the metabolic needs of different tissues, cardiac activity, and respiratory procedures such as gas exchange. This coupling will be achieved by the autonomous nervous control system.^[23] More developmental changes include humoral and nervous control mechanisms, which will actually combine so that response to physiological and environmental changes will be met with a more coordinated reaction. The nervous system assists in development of cardiovascular functions, but this change will occur much later in life.^[23] Any time there is a switch from one type of respiration (e.g. cutaneous) to another (e.g. lung) there will be a required alteration and modification of the circulatory system. Another increase that comes with development is that of blood pressure. This is due to both the increase of blood in the organism due to size increasing, and as a response to the enlargement of internal organs.^[23]

It has been said earlier that T. carnifex is an oxyregulator, and internally can change its physiology to maintain respiration even in oxygen poor environments. However, this is not true for the larval and embryonic stages of T. carnifex. At these points in development, the T. carnifex are oxyconformers. In the earliest stages of embryonic life, cardiovascular activity is not linked or correlated with the activities that produce metabolism changes as in adults.^[23] Thus, many changes will have to occur for the organism to reach the mechanisms it contains as a fully developed animal.

Interestingly, the heart (and related structures) is the first organ to operate functionally. This is necessary for many reasons, as the heart is responsible for distribution of countless things throughout the body and embryonic development will obviously require a great deal of things to proceed normally. One such reason of its primary operation is the structuring of the vascular system, such as capillary networks found in varying tissues.^[23]

Ontogenic development

Ontogeny of mechanisms in T. carnifex is vital in understanding how the organism will function as a whole. This includes the development of the organism, and why and how it develops in the manner that it does.

Many things will increase during development from embryo to adult. The main changes are blood pressure increase, an increase in the amount of blood one cardiac output (cycle) will produce, and an increase of stroke volume.^[25] There are not only increases seen during this time. Peripheral resistance (resistance to blood flow found in peripheral venous systems) will actually go down, allowing the circulation of the organism to become more facilitated.^[25] Development can be shown by T. carnifex’s response to hypoxia. This response will demonstrate the extent to which neurohormonal instruments are at work operating in the cardiovascular system. Blood pressure in response to hypoxia will go up in adults, but will not follow this pattern in younger organisms. In adulthood, this extent will be much larger in comparison to juveniles.^[25] The cellular (myocyte) membrane will alter its permeability as adulthood is reached, adrenergenic/cholinergic systems will develop, and the gas transport mechanisms in the formation and modification of ion channels will also develop. In early development, it is possible (but not proven) that cardiac receptors have an influence on the regulation of blood pressure.^[25]

Variability in the cardiovascular system is often due to hormonal changes, or the innervation within the system.^[25] Nerves and the nervous system have a great deal to do with the activity of the heart and the functions that it will be performing at any given time.

Osmoregulation

Physiological challenges faced by T. carnifex

T. carnifex’s physiological response to nonylphenol

One organ that is extremely important in the physiology of the Italian crested newt is the adrenal gland, because it plays a vital, if not the most important, role in stress response.^[26] This gland controls the release of corticosteroids and catecholamines – such as corticosterone and aldosterone – which play the primary role in the regulation of water and ion balance.^[26] To allow survival in the environment as well as adaptation, the hormones must work to eliminate or neutralize stimulus that is stressful to the animal.^[26] They do this through induction to changes in metabolism and ion regulation.^[26]

Some of these stressors and stimulus that must be neutralized come from the environment of the Italian crested newt. T. carnifex lives in an open aquatic environment relatively close to human activity, which makes it prone to sewage leakages. It is noted that the absorption of the direct environment is a daily part of T. carnifex's life. This being said, it is clear that should there be toxins in the aquatic environment of the newt, it is highly likely and probably guaranteed that they will be absorbed into the body of T. carnifex. Chemicals such as nonlyphenol and ethoxylate, which are endocrine disruptors, are two of many found in the newts' environments, and are considered the most toxic when transformed by microorganisms.^[26] This presents a very clear and present problem, because once these chemicals enter the skin, they become integrated into the newt’s bodily functions and can cause great harm to the animal. Because T. carnifex osmoregulates mostly through transepithelial movement of water and solutes, this is probably the largest physiological challenge faced by the newt.

Nonylphenol is a large issue for T. carnifex because it is a very common toxin found in leakage from sewers into the habitat of the Italian crested newt. Newts are amphibians, and make their habitats in aquatic environments. These environments have been known to sometimes receive the previously discussed sewage leaking. The compounds nonylphenol as well as ethoxylate surfactants can be found in polluted rivers, where the newts reside. The two substances discussed can be transformed, by microorganisms, into compounds which are much more toxic.^[26] They are extremely good bioindicators, due to the magnification of these chemicals in the environment demonstrated in the newt. Nonylphenol absorbed through the newts’ skin is especially dangerous because it can result in issues with the reproductive biology as well as cutaneous and osmoregulatory mechanisms.^[26]

Besides mitochondria, there is a multitude of lipid droplets in the cytoplasm. Lipid droplets assist in the movement of substances across the integument.^[26] Another negative effect of the chemical nonylpheno, a decrease in the presence of lipid droplets, can be observed in the cytoplasm.^[26] These are vital to the level of permeability that the skin has.

Steroidogenic cells found in the Italian crested newt produce corticosteroids. Corticosteroids are corticosterone and aldosterone. These have different quantities according to the time of year. Corticosterone is very low in October to November, then peaks in January. Levels lower again in March and then peak in July. Aldosterone, on the other hand, has differing levels.^[26] Levels are low in September to November, and rise to a maximum from December to April. Then there is a steady decrease lasting from May until July. In experiments allowing newts to be exposed to nonylphenol, there was a decrease in corticosterone and aldosterone.^[26] This is because of the direct effect that nonylphenol has on the adrenal gland, which is a reduction of the production of both corticosteroids. Both steroidogenic and chromaffin tissues have an effect on them deriving from the exposure. This will lead to amphibian decline.^[26]

There are other chemicals that will lead to osmoregulatory issues within the physiology of the newt, and broader spectrum factors that may not affect osmoregulation specifically but will cause problems within the newt’s life cycle. These include things like destruction of habitat and predators. But the most prominent issue is the poisoning and polluting that occurs in the newts' habitat, causing them to inadvertently absorb harmful substances into their body.

Newt reactions to altering salinities

Because T. carnifex is a freshwater animal, and found mostly in rivers, it is unlikely and probably impossible that it will ever be naturally found in a marine environment. That being said, it is possible that experimentally this situation could arise. In this hypothetical environment, similar mechanisms to marine fish would be employed. To achieve equilibrium between internal and external environments, the newt would likely be inclined to absorb salt through the integument and/or ingest saline-rich substances orally.^[16] In marine animals, water uptake is mainly driven by sodium and chloride absorption, and it may be assumed that if T. carnifex were to be placed into a marine environment, they would undergo similar adaptations.

Because they are trying to reach equilibrium with a salt free environment, there will be mainly an outwards flux of salts into the environment while attempting to match their internal self with the saline-free external environment.^[16]

Water loss versus water gain

The direct environment the newt finds itself in will be the most important factor in whether there is a loss or gain of water through cutaneous absorption. The main idea here is that T. carnifex will be attempting to equate its internal solute concentration with that of its environment. Factors such as salinity, ion movement, and hormonal trigger will account for whether the newt will undergo a loss or gain of water.^[16]

There will be a variation in water loss and gain between newts in the aquatic, or winter phase, and the terrestrial, or summer phase. In the terrestrial environment, there will be a change to the integument that will directly affect osmoregulatory mechanisms. This change is the shift from a smooth, mucosal skin surface to one that is rough and dry. This change is to allow easier transport of water through the cutaneous membrane and into the newt to allow for hydration.^[27] A mucosal surface is difficult for substances to get through, and is generally attributed to an animal to prevent transport across the epithelium.

In experiments, it was discovered that dehydrated newts were prone to a loss of motor control. After only 22% water weight loss, newts in the aquatic phase lost their ability to remain upright and mobile. However, after adaptation to a terrestrial phase, they could lose 30% before a loss of motor control was recorded. This is an indication of how vital water levels are to the function of T. carnifex.^[27]

Newts in the terrestrial phase were found to dehydrate much quicker than newts in the aquatic phase, but conversely, during rehydration, dehydrated terrestrial animals will go through water gain 5x faster than dehydrated newts that are in the aquatic phase.^[27]

Ion uptake will also show a change between the two phases. Obviously, water loss or gain and ion transport go hand in hand when discussing osmoregulation in T. carnifex. Terrestrial phase newts will show a near doubling of sodium movement into the newt, which is said to be unidirectional.^[27] This large uptake in correlation with the terrestrial phase will indirectly increase the transport of water across the epithelium.

Newts in the aquatic phase will undergo a much higher amount of water gain, which will cause an increase in urine output. Also, aquatic newts will undergo a loss of ions through diffusion across the surface of the body. Terrestrial phase newts, on the other hand, face an alternate issue with water loss. This is explained by the increase in sodium uptake.^[27]

Direction of diffusion/osmosis between environment and organism

One important equation to apply to the ion and water movement into T. canifex is that of Hertz’s convection-diffusion equation. The lateral intercellular space (lis) is a main player in this equation, and the equation itself is what is applied to determine many things about the leaving of water together with solutes from lis.^[28]

Experimentally, it was discovered that a lowering of fluid absorption from external environments is shown in the presence of serosal bumetamide.^[28] It was also discovered that there is a distinct relationship between the solute concentration deemed necessary to decrease transepithelial flow, and hydraulic permeability.^[28]

There is one structure, aquaporin, that above all else proves most significant for the water transport across a membrane. Even though osmosis is being mainly discussed, this is just one of a few tactics for transporting water. These other tactics being referred to are known as isosmotic transport.^[28] This can be described as water being able to flow in bulk minus the motivating epithelial forces. Isosmotic transport is, however, dependent on the active transport of sodium.^[28]

Another important equation to assist in determination of values having to do with osmoregulation is Heff’s equation. This describes the tie that exists between the osmotic pressure of a diluted solution and the concentration of the solutes that are dissolved in said solution.^[28] This equation is as follows:

                                                    Π=RTCS

A few other equations to assist with determining values in osmoregulation are volume flow per unit membrane:

                                                    JV=LPRTơSΔCS

And the membrane’s osmotic permeability:

                                                     Pf=RT x Lp
                                                           Vw[28]

It is important to realise that a very small change in hydrostatic pressure can create a significant motivating force for the movement and transport of water through and across the membrane. Osmosis of substances happens to the kidney’s proximal tube, the small intestine, skin, and glands.^[28] Na+ pumps create a flux of sodium into the lis. This is reasonable considering that there is a large number of sodium pumps on the lateral membranes of transporting epithelia. Once the pumps are put to use, the lateral intercellular space (lis) will become hyperosmotic with respect to the external bath or environment. When equilibrium is reached at the transepithelial equilibrium, the water flow will be lumen into blood, according to Curran.^[28]

Curran says that the correction of water flow in skin can be attributed to altering reflection coefficients that exist at the opposing boundaries. This is what will couple water flow-in across the tight junction with active solute flow-in. In this system, this will mean that the rate of water influx is directly proportional to the amount of Na+ going into the system (E. Larson et al. 2007). If the sodium pump enters lis enough so that there is sufficient concentration, then water may move osmositcally across an adverse transepithelial osmotic gradient. Again, the Hertz convection-diffusion equation says much about the entering of water and solutes across a membrane. Na+ is transported through principle cells, and Cl- is transported through mitochondria-rich cells.^[28]

The Diamond-Bossert theory is a predominant model in terms of isosmotic transport. It speaks to a concentration gradient of 0 of the solute concentration gradient across the lateral intercellular space.^[28] Fick’s Law is of vital importance in explaining this model with regards to diffusion. The model explains that if sodium pumps can be confined by the closed end of the lis, with particular values inhabiting the parameters that characterize a very specific system, then the constant can be zero and equilibrium will have been reached.^[28] The parameters in question are length and radius of the space, water permeability of the lateral membrane, inflow of solute, and the diffusion coefficient of the solute. A necessary assumption of this model is that the tight junctions will not allow water to pass without the coupling of a solute.^[28]

Another model is that of electro-osmosis. This theory harbours the idea that electro-osmotic coupling of Na+ and water is what determines the secretion or absorption of salt and water. Electroneutrality is critical here, and this can be maintained through anion flow throughout the involved cells.^[28]

A last model of osmotic flow in T. carnifex is osmosensor feedback. This explores the probability that aquaporins are the regulating force and that there is a protein in the walls of these that senses of transmembrane osmotic gradients (Hill and Shachar-Hill experiments). There is an assumption here that the cellular exit of ions that are actively transported are confined exclusively to the basal plasma membrane.^[28]

There are two major factors that categorize epithelia that transport fluids. These are vital to determine which organs are doing what in an organism. These features are the transport that occurs when no external driving force appears, and the uphill transport of water.^[28]

To sum up, sodium/potassium pumps can be found on the lateral membranes of fluid transport epithelia. There will be fluid in the lateral space that during osmosis will become hypoerosmotic with regards to the external environment.^[28] The Hertz convection-diffusion is instrumental in determining a direct analysis of the coupling between water and solute uptake. For all the theories, it must be assumed that the activity of sodium uptake is purposely regulated to satisfy the requirements of isosmotic transport. It is more than likely that epithelial cells contain osmotic sensors.^[28]

Description of the anatomy and biochemistry involved; The skin

Anatomy

The skin of T. carnifex is a highly permeable structure, because it is the source of osmoregulation.^[29] Due to their skins' ability to absorb water, these newts do not need to drink.^[29] T. carnifex’s epidermis is divided into three separate cell layers: the stratum corneum, the outer layer, which consists of keratinized cells, the stratum intermedium, the middle layer, which consists of polyhedral cells, and the stratum germinativum, the basal side layer, which consists of a single row of cells.^[29] The first layer of cells contains three adaptive characteristics that help protect the epidermal layers.^[29] The inner portion of the integument consists of the connective tissue that makes up the dermis layers. This includes the stratum spongiosum, which is made up of loose tissue containing collagen, elastic fibers, blood vessels, glanular and muscous glands, and the stratum compactum, which consists of dense collegen fibers in bundles lying on the inside. The stratum spongiosum is closer to the surface.^[29] Altogether, this layer measures 5 micrometers. The thickness and resistance of this layer, combined with the filamentous network of keratin, interfilamentous intermatrix, and the intercellular substance produced by the kerinocytes all help to reduce chemical and physical damage to the underlying tissues and all help to keep a constant internal environment.^[29] However, water is still able to get through the layers of the epidermis.^[29] Keratinocytes are especially important to the newt’s physiological well-being because they are responsible for the absorption of sodium ions.^[30] When it comes time to shed the skin, these cells will thicken and flatten out and eventually become the skin the newt sheds.^[30] Since highly permeable skin is a favourable adaption this is the main source of osmoregulation in crested newts.^[31]

Physically, skin also helps to limit water loss by the mucus and the intercellular spaces in the stratum corneum.^[29] The permeability of the skin varies depending on what part of the body the skin is on, as well as how smooth the skin is. The smoother the skin, the less water is absorbed.^[29] One type of cell that is thought to play a role in the osmoregulation of amphibians are flask cells, which are present in amphibians' skin and reside just below the stratum corneum. They contain a bulging basal portion with a thin apical neck, and when stimulated in some species, separates the stratum corneum from the other epidermal layers, further decreasing water loss in terrestrial phases (Toledo and Jared 1993). Skin thickness is also related to the rate of water loss and uptake; the thicker the skin, the slower the uptake and loss.^[29] An advantage of having thicker skin is that it helps prevent the organism from suffering an excessive loss of water.^[29] A high degree of vascularization, cutaneous lipids, and morphological adaptations can all serve to limit water loss and also increase rehydration once the organism comes back into contact with an aquatic environment.^[29]

Anatomical adaptations

Salamanders, a relative of the newt, have distinct grooves arranged along the ventral surface to the dorsal median area, allowing for more water to be absorbed through the skin.^[29] Costal grooves exist along the body as well and their function is to deliver water to the areas on the body that do not receive any type of contact with water.^[29] The costal grooves between the ribs are more developed allowing for water transport in areas of the skin that does not come in contact with water.^[32]

Hormone function and involvement

This adaptation appears to be more favourable when the newt is living in a terrestrial environment because the newt needs to take advantage of all the water possible in order to maintain a healthy ionic balance.^[31] This permeability is controlled by hormones from the pituitary gland, which include vasotocin, prolactin, and thyroidal, which all help in sustaining life in a terrestrial environment.^[31] This is especially helpful since the crested newt goes through changes in its environment, depending on the season.^[31] During the winter, when it goes through its breeding stage, it mostly lives in an aquatic environment, where it does not have to produce an abundance of these hormones as water is readily available and Na+ transport has been discouraged.^[31] One possible explanation is that water is not moving down its concentration gradient and the absorbed volume is being controlled by renal filtration. The complexity of the osmoregulatory system has, as of yet, not fully understood by biologists because of T. carnifex’s seasonal hiding behaviour.^[31] However, in the experiment done by Lodi et al. some of these mysteries were solved.^[31] Their observations showed that breeding phase animals in their aquatic environment had exceptional low permeability which tended to minimize the dilutions of the inner fluids.^[31] High permeability was observed when in the terrestrial environment, which caused rapid rehydration.^[31] There is a suggestion that osmoregulation can be modified by hormonal or environmental factors.^[33] This places the skin at great importance when accessing osmoregulation.^[31]

Climate/season and hormone duality

Temperature plays a key role in the osmoregulation of the newt. Not only do the changing seasons drive T. carnifex either into the water or onto land, it also regulates the release of certain hormones in accordance with the osmotic permeability of the newts' epidermis. Osmotic permeability is dependent on the release of hormones such as prolactin, vasotocin, as well as other thyroidal and internal hormones.^[34] Brown et al. experimented on a close relative of T. carnifex to determine the effects different environmental conditions have on the newts osmoregulatory systems.

Prolactin is one of the major hormones released with the changing temperatures, and as such, plays an important role in the transport of sodium and water ions. An increase of this hormone results in the decreased permeability of the skin as well as a reduction of active sodium uptake, which seems to play a less important role than previously thought. This becomes apparent when newts are in their aquatic environment.^[31] During winter months, prolactin is released into the circulatory system, which drives T. carnifex into the aquatic environment (Lodi 1981). Not only does this hormone prompt a change of habitat for the newt, it reduces the active transport of sodium ions.^[34] This happens because there is more water readily available to the newt for uptake as compared to its terrestrial dwelling during the summer months.^[35]

Arginine, vasotocin, and adrenocorticotropic are hormones secreted by the adrenal glands and are a key component in a newt's regulation of water.^[35] In contrast to prolactin, which decreases osmotic permeability, vasotocin increases the permeability and is secreted during the summer months.^[35] This hormone is regulated by another major hormone – adrenocorticotropic hormone (ACTH). Arginine vasotocin not only increases cutaneous water permeability, but promotes increased cutaneous blood flow.^[36]

Response to seasonal changes

Changing the environment from a terrestrial to an aquatic one will create a reduction of urine flow and production, which also results in a decrease in glomerular filtration rate (GFR). In these situations, kidney functions associated with water reabsorption do not make a large impact on the reduced output of urine.^[37] In winter, when the newt is in the aquatic phase, the skin’s osmotic permeability is highly reduced. This can be measured by observing a reduction in urine output.^[37] Kidney function can also increase urine production by reducing GFR with no change to tubular water reabsorption, or by increasing water reabsorption coupled with a reduction in GFR.^[37] The terrestrial phase is characterised by an upkeep in high GFR and urine flow.^[37] This maintenance is associated with an increase in the permeability of the integument, which allows for quick re-hydration.^[37]

Ion and solute transport and effects

The transepithelial potential – or TEP – of a newt's cell is upheld by the intake of sodium in vivo.^[35] Brown et al. performed an experiment to observe the sodium permeability and saturation effect. What was discovered was that when the external concentration of sodium exceeds 10-20 mM within the presence of the Cl anion, the TEP decreases.^[35] A plateau is reached for the concentration of sodium inside the cell when the external concentration is greater than or equal to 10mM in the presence of the sulfate ion.^[35] These data suggest that the saturation of the sodium transport mechanism occurs at around 10mM.^[35] The slightly different results shown in the presence of differing anions has been interpreted as being reflective of integumental permeability, or leak conductance, of sodium at high temperatures.^[35]

Alkaline phosphatase in cooperation with ionic transport

Alkaline phosphatase, an enzyme located in the proximal tubules, plays an important role in the polarization of membrane surfaces.^[33] While the activity of this enzyme varies across seasons and the mating habits of the newt, the exact role this enzyme plays is not particularly well understood.^[33] From the experiment performed by Dore et al., it was observed that the alkaline phosphatase activity was reduced in the transport of two anti-diuretic hormones, prolactin and arginine-vasotocin. It was also noted that the activity of alkaline phosphatase decreased, due in part to hypophysectomy.^[33] In summer, aldosterone creates an increase in enzymatic activity (Lodi et al. 1995). Because of this increase in activity, there is a decrease in ion transport, which ties into the activity of ion transport during the aquatic phase.^[38] This is likely due to the lowering of bodily functions. Of course, there is an involvement of APH activity in the mechanisms involved in ion transport across the epithelial membrane, also called the cutaneous epithelium.^[38] Epithelial cells undergo frequent renewal, and this, in combination with the processes that lead to the rapid regulation of membrane transport, will lead to the said regulation.^[38] The APH molecule, in short, plays a large role in the ion exchange across the membrane between the internal and external environments (Lodi et al. 1995). Aldosterone, will independently cause an increase in Na+ channels within the cutaneous membrane.^[38] The ion transport is a direct result of coordination and cooperation of both MR cells and the principal cells; for instance, a main role of the mitochondria-rich cells is to energize sodium absorption through use of the principal cells.

Special adaptations of T. carnifex

Newts of many different species are often found congregating in ponds, therefore niche overlap is very common.^[39] T. carnifex has adapted a specialized niche, to avoid species competition. Fasola (1993) discovered that during reproduction, water depth is the most important factor, and to avoid competition T. carnifex reproduces in deeper waters. Compared to most adult newts, T. carnifex is significantly more nocturnal and this helps to reduce resource competition between other newts who are more active during the day.^[39] During metamorphosis, T. carnifex becomes roughly eleven times heavier than other smaller newt species in a specific pond.^[40] The weight of T. carnifex allows it to prey on other species of newts, and therefore allows for an increase in its survival rate.^[40] Buskirk (2007) found that when T. carnifex was exposed to smaller species of newts, such as T. helveticus, they caused delayed metamorphosis and decreased fitness in T. helveticus. Conversely, when exposed to a newt species that had a larger body mass, T. carnifex did not affect metamorphosis or fitness in the opposite newt.^[40] Buskirk proposed that body size of Triturus newts plays a large role in the determination of species interactions in a natural wetland environment. Due to the larger body size of T. carnifex, positive interactions with other smaller newt species is limited and allows for more resources and environment for T. carnifex.^[40]

During times of stress, adrenaline, noradrenaline, and dopamine are excreted from chromaffin cells located on the ventral surface of the adrenal glands.^[41] Generally these hormones are secreted from two types of chromaffin cells, but in T. carnifex they are only secreted from one type of chromaffin cell.^[41] The secretion of these hormones in T. carnifex is dependent on the temperature of the environment; in winter and summer more noradrenaline than adrenaline is present, but in spring and autumn the hormones appear in almost equal amounts.^[41] Perry and Capaldo (2011) discovered that chromaffin cells in T. carnifex can also be affected by many hormones, including ACTH, FSH, and acetylcholine by increasing the secretion of adrenaline and noradrenaline.

Respiration in newts can take place through the skin, the lungs, or the buccal cavity, but newts mainly respire through the skin due to the fact that the buccal cavity and lungs are poorly vascularized.^[42] However, when T. carnifex are placed in poorly oxygenized water, they switch their primary respiratory surface from skin to lungs.^[42] Through further tests it was shown that T. carnifex can stay under water for long periods of time by using their skin to respire, but when they become active they must switch and usetilize their lungs or buccal cavity.^[42] Eddy and McDonald (1977) also discovered that many nsetilize their lungs to help regulate their buoyancy, similar to fish that use a swim bladder to aid in swimming.

Thermoregulation and metabolism

The Italian crested newt is a poikilothermic ectotherm. This means that they do not thermoregulate by internal forces, but rather adopt the temperature of the surrounding conditions. This can be either advantageous or problematic, depending on the situation. Because of the phase-changes of T. carnifex, there will be a large variation in the temperature of their surrounding environments. This means that it is necessary to use mainly behavioral mechanisms to survive in the changing temperature of their environments.

The Italian crested newt can be classified as ectotherms due to their inability to internally regulate their temperature. The body temperature of T. carnifex is dependent on the temperature of the external environment.^[43] Because of this, they can be said to lack the mechanisms necessary for physiological regulation of temperature, and must instead rely on behavioral thermoregulation. An example of this is the selection of microenvironments to keep their body temperature within a “preferred range” (the temperature at which their body functions are running optimally).^[43] Even though this does assist with short-term fluctuations, there is still the issue of seasonal climactic changes, which will persist for much longer. The internal temperature of the newt will affect the rate of the chemical reactions that take place inside and within the animal’s physiology.^[43]

Behavioral thermoregulation

A larger body size will be the result of colder temperatures; the Italian crested newt can actually increase its body size in response to a decrease in temperature.^[44] There have been studies that show that there will be a greater increase in body size in females.^[44]

T. carnifex are ectotherms. This means that the internal forces of this newt that control the body temperature have almost no importance.^[45] They are traditionally called “cold-blooded” although blood has really nothing to do with how these animals thermoregulate. This means that T. carnifex will regulate in a way that depends more on behavior.^[45] When there is a large range of environmental temperatures, newts are very insensitive to a thermal gradient profile.^[45]

Thermoregulation, in combination with seasonal acclimation, describes the major mechanisms of how ectotherms (T. carnifex included) cope with the changing temperatures existing in their environments. This regulation is most often achieved through behavioral thermoregulation.^[46] They are thermoconformers, which means they will acclimate to their surrounding environmental temperatures.^[46] Because of this, there is an increase in the time allotted for thermoregulation.

Development and morphological changes and thermoregulation

Since predators are an issue for T. carnifex, there will be a vulnerability associated with their presence. The larvae of newts are extremely vulnerable as they cannot defend themselves. In order to protect against these predators, larvae will actually shift their microhabitat to a temperature range that exists outside the predator’s preferred temperature range.^[47] This defense mechanism basically means that newts in more vulnerable stages will employ the defense mechanism of existing at temperatures outside the range of their predators. The growth and development of these larvae will be largely dependent on these temperatures.^[47]

While newts are developing, they go through a stage of metamorphosis. Larvae that are in the metamorphosizing stage tend to prefer warmer temperatures than those in the stage following metamorphosis.^[47] Therefore, the larvae in this stage will undergo a much more precise thermoregulation process than those in the intermediate stage.^[47]

Since T. carnifex are amphibians, they must undergo changes in morphology that allow movement from the aquatic phase to the terrestrial phase. These changes are associated with alterations in the thermal environment.^[48] The thermal environment of the terrestrial phase does produce many more daily fluctuations of temperature change than does the aquatic phase.^[48]

Reproduction and thermoregulation

T. carnifex females will undergo reproductive processes. Many processes, such as digestion and locomotor activity, will result in a change in thermoregulation, specifically higher preferred body temperatures. Reproduction is one of these thermogulation-changing processes.^[49] Reproductive females often exist at a preferred body temperature of 2.3-4.3 degrees Celsius higher than both those of non-reproductive females as well as males. This is true regardless of the activity level of the animal in question. There is a much narrower range of body temperatures in reproductive females, which means that there is much more precision in thermoregulation.^[49] There are different preferences for reproducing females, most likely because there is a very specific temperature that is optimal for embryonic development of T. carnifex. An upward shift in the maternal body temperature will increase the rate of development. This temperature will influence a number of things: survival, body size, growth rate, and locomotor performance.^[49] Embryonic development is very important on the temperature of the surrounding water. When faced with a thermal gradient, reproducing females will show a preference for higher temperatures. Preferred environmental temperatures are approximately 10-24 degrees Celsius from March to November, and 4-8 degrees Celsius from November to March.^[49] Body temperatures of T. carnifex will closely resemble those of the surrounding environment. This change will often occur within 1 minute. It is likely that this is all because estrogen induces a shift in the preferred body temperature.^[49]

Dealing with thermoregulatory physiological challenges

T. carnifex inhabits an aquatic environment much of the time, and this particular environment presents many thermoregulatory challenges.^[46] This is because of the seasonal variation. To deal with this, the Italian crested newt will use both thermoregulation and thermo-acclimation.^[46] The external environment is heterogeneous and must be dealt with in three ways: thermoregulation, thermal acclimation, and thermal sensitivity.^[46] The co-adaptational theory suggests that there is a combination of behavior and physiological compensations. This theory is based on the costs and benefits of thermoregulation, and hypothesizes that as the costs of thermoregulation go up (things like energy and time), the precision of thermoregulatory activities will decrease and relax.^[46] This response will therefore change the benefit that is received from a particular body temperature. This is why T. carnifex will conform to the external temperature, instead of regulating internally.^[46]

In the laboratory, the costs of thermoregulation are lowered. In these circumstances, the maintenance of body temperature will exist in a narrower range. This indicates to researchers what temperatures the newt attempts to achieve and maintain in its natural environment as opposed to a lab.^[46] Ectotherms can modify their preferred body temperatures in response to the temperature change that comes with the altering seasons.^[46]

The temperature will determine the equilibrium that is reached between the formation and disruption of factors (ex. noncovalent bonds) that steady and stabilize cellular membranes (and other biological membranes) and the high-level structures of proteins that exist within T. carnifex.^[43] Structural flexibility is a requirement of functionality for the fluid mosaic membrane that exists in animal cells. On top of this, temperature also determines the degree to which enzymes can act as catalysts.^[43]

With this in mind, we can understand how a drop in temperature will decrease the fluidity and therefore the efficiency of cellular membranes in T. carnifex. This will pose a challenge for the Italian crested newt when temperatures begin to drop, and can be applied to the broader category of challenges faced by T. carnifex with respect to thermoregulation.^[43]

There is a scientific idea, known as temperature compensation of metabolism, that refers to the maintenance of similar and stable metabolic rates in the face of varying temperatures. T. carnifex undergoes attempts in this domain to achieve this idea.^[43]

A systematic state in T. carnifex will refer to multiple things going on in the physiology of the animal. This can be things like ionic distribution, alternative enzyme patterns, and differing metabolic patterns.^[50] At a specific temperature, a state can be achieved that can be called as “steady”. More defining factors are the specific concentrations, or turnover rates of bodily components. These components can be anything ranging from ionic concentration to the fluidity of the membrane.^[50]

A fall in temperature can result in a decrease in the number of molecules that are available within the organism per unit of time. A drop in temperature will lower this number and physiological functions can come to a standstill.^[50] This is how an extreme drop in temperature can result in the eventual collapse of physiological functioning and eventual death of an animal. Ectotherms are unable to survive in extremely cold temperatures (around 0.7 degrees Celsius) as their internal body temperature must match the outside temperature and cannot handle very cold temperatures.^[50] However, this can be dealt with in a few different ways, and one such way is the changing of actual lipid conformation and composition. This adaptation will cause a rise in fluidity and a higher physical tolerance of the cells undergoing this temperature change.^[16]

On the other hand, rises in temperature will likely result in the eventual denaturation of proteins within T. carnifex. This is obviously an extreme problem, but even before that problems will arise. Before denaturation, a rise in temperature will lead to an increase in permeability of the biological membranes.^[50] This my seem minor, but the delicate balance of unequal distribution of ions and small molecules on either side of the membrane will not be maintained.^[50] This is extremely dangerous for the functioning of T. carnifex.

Interaction of metabolism and thermoregulation

This response to changes in temperature (mentioned above) is related to metabolism in the sense that limitations will be placed on the extent to which ectotherms’ metabolism can change. Altering temperatures produce limits on the lengths to which metabolism has the ability to change.^[50]

With regards to metabolism, a higher rate of thermoregulation means an increase in metabolic rate, and therefore a higher consumption of oxygen.^[49] Standard metabolic rate will alter as the ambient temperature alters.

Applicable laws and principles to thermoregulation and metabolism

In poikilotherms, there are a number of laws that are followed on the basis that these organisms adjust their internal body temperature to conform to the external environmental temperature. One such basic law is that any change in Tb will be directly proportional to Ta.

Two other laws have more to do with the metabolic rate:

• The metabolic rate will increase in relation to Tb, and
• The metabolic rate is exponentially related to Tb.^[16]

Description of adaptations involved in temperature regulation

Behavioral avoidance or changes

Thermoregulation in T. carnifex can be affected by both metabolic and behavioral changes.^[51] Aquatic ectotherms will use behavioral adaptations to compensate for the wide variety in temperatures within their environments.^[51] Newts often select the temperature they prefer at a certain time of day in order to control their body temperatures.^[51] In the larval stage it is important for the growth of the individual to optimize the temperature that allows them to get the most benefits physiologically, and so they will live in the temperature that allows for the greatest development.^[51] Adult newts sometimes dive for food and reproduce in the water, so they will dive to the optimal temperature that requires the least amount of variation in their body temperature.^[52] By reducing the amount of variation that the newt's body faces, it will reduce the metabolic cost of the organism, and allow energy to be used elsewhere.^[52]

Wu et al. (2007) found that when larvae are placed in a homogeneous temperature environment, they do not need to use behavioral thermoregulation and instead only use metabolic compensation. However, when placed in an environment with a variation of temperatures, newts will use both behavioral thermoregulation and metabolic compensation.^[51] Samajov and Gvozdik (2009) discovered that when adult newts dive at higher temperatures, they reduce the amount of time in their dive in order to reduce the cost on thermoregulation. Samajov and Gvozdik did not take into consideration that newts respire aerially, as well as cutaneously, and so the frequency and the duration of the dives recorded could have been affected by the amount of dissolved oxygen in the water. Adult newts alter the speed in which they dive between descents and ascents.^[52] Descents affect thermoregulation by using more energy, and so newts will often choose a passive descent, rather than swimming down to forage for food.^[52] When T. carnifex ascends, thermoregulation is not as affected because newts have been known to increase their buoyancy by expelling excess gasses.^[52] By altering the behavior used as a larvae and when swimming, T. carnifex can effectively thermoregulate.

Physiological changes to blood flow or respiration that alter heat exchange

Due to the nature of this newt’s secretative behaviour and most of the research being on their behavioural thermoregulation, there has not been much research on the metabolic interactions between respiration, blood flow and thermoregulation. However, in general newts are more subject to higher oxygen consumption during diving, which puts a strain on reproductive success and ultimately reduces metabolic activity.^[53] Newts are also greatly restricted in metabolic activities due to their anaerobic lifestyles, and because of this newts do not seem to meet their metabolic threshold. This cannot be fully determined due to their secretive lifestyle.

Specialized organs or anatomy that relate to heat exchange

Amphibians and reptiles are poikilotherms, and as such are dependent upon external heat sources to regulate their body temperature, and unlike homeotherms do not actively maintain a constant internal body temperature.^[54] The main organ involved in amphibians’ heat loss and gain from the environment is their skin (Shoemaker, VH). Newts keep their integument permanently moist, and transitioning between terrestrial and aquatic habitats with such a good conductor of heat allows them to maintain consistent metabolism in a variety of ambient temperatures.^[54] In one particular field study, it was found that water at the edge of a pond varied by as much as 6.5 °C in one day.^[54]

Regulation of metabolism related to temperature regulation

Temperature in amphibians and reptiles directly affects metabolism in an exponential manner, and among ectotherms, temperature is one of the most important factors, acting directly upon metabolism.^[55] Since the newt is a poikilotherm, the purpose of this increase in metabolism is not to generate heat to regulate their internal temperature, but to counteract the depressive effect on standard metabolism that low atmospheric temperatures produce.^[56] For example, there is a direct correlation between the activity of certain muscle metabolic enzymes and the preferred body temperature of Eastern red spotted newts.^[54]

In general, thermal and exercise physiology are not independent traits in amphibians; however, there is much variation from species to species and is a direct result of each species’ activity patterns and behaviours (Navas et al., 2007). Amphibians have a range of temperatures in which their locomotive performance and metabolism is optimal, as well as an ability to adjust their performance-to-temperature curve in response to a wide variety of atmospheric thermal environments (Navas et al., 2007).

Like homeotherms, poikilotherms and ectotherms usually exhibit a thermal preference, although not quite in the same way. Many fundamental aspects of thermal biology in amphibians and anurans are poorly known or biased towards a few species, although several studies do exist to provide a general overview of the responses and causes (Navas, Gomes, Carvalho. 2007). While homeotherms have a thermal-neutral zone (TNZ) in which no extra metabolic energy is spent on the regulation of body temperature, amphibians have a zone in which their metabolic processes are at their optimum (Navas, Gomes, Carvalho. 2007). Expenditure of extra metabolic energy in amphibians when body temperature reaches a lower limit occurs only to prevent a reduction in metabolic rate caused by these lower temperatures (Navas et al., 2007).

Oxygen consumption has normally been determined to be the most convenient and relevant measure of metabolic activity. With very few exceptions, oxygen consumption is reduced in response to starvation and physical inactivity, and increased in fed and active animals (Hawkins, 1995). Basal maintenance metabolism in ectotherms is relatively unchanged in response to variations in atmospheric temperatures, which would be advantageous to the newt’s survival in both their aquatic and terrestrial environments where temperatures can rapidly shift throughout the year.^[55]

Special adaptations unique to habitat and lifestyle and/or integration of systems

T. carnifex faces an interesting challenge, one that all air-breathing aquatic newts must face: the temperature variance between the land phase and the aquatic phase. Since water has a heat capacity 3500 times greater and a thermal conductance 23 times greater than air, small ectotherms such as the Italian crested newt are likelier to have a more variable body temperature than strictly terrestrial organisms.^[57] Many ectotherms possess the ability to reversibly alter their preferred body temperature based upon the seasonal changes the newt experiences, which means that their optimal thermal range is quite wide.^[46] They respond to variance in temperature by employing either behavioural or physiological changes. Some changes in body temperature are unavoidable, however, depending on how much solar radiation is available.^[58]

It has been debated whether a behavioral thermal adaptation is actually advantageous. In fact, some have decreed it to be an evolutionary inhibitor.^[47] Because the newt must use solar radiation to help regulate its body's temperature, it leaves them exposed to more predation, especially when it is still in its larval stage.^[47] While much speculation has been made for how preferred body temperature acclimation works, its significance has been mostly unexplored.^[46]

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External links

• Media related to Triturus carnifex at Wikimedia Commons
• Data related to Triturus carnifex at Wikispecies