Distribution of FMRFamide-like immunoreactivity in the nervous system of the slug Limax maximus

Summary The distribution of FMRFamide-like immunoreactive neurons in the nervous system of the slug Limax maximus was studied using immunohistochemical methods. Approximately one thousand FMRFamide-like immunoreactive cell bodies were found in the central nervous system. Ranging between 15 m and 200 m in diameter, they were found in all 11 ganglia of the central nervous system. FMRFamide-like immunoreactive cell bodies were also found at peripheral locations on buccal nerve roots. FMRFamide-like immunoreactive nerve fibres were present in peripheral nerve roots and were distributed extensively throughout the neuropil and cell body regions of the central ganglia. They were also present in the connective tissue of the perineurium, forming an extensive network of varicose fibres. The large number, extensive distribution and great range in size of FMRFamide-like immunoreactive cell bodies and the wide distribution of immunoreactive fibres suggest that FMRFamide-like peptides might serve several different functions in the nervous system of the slug.     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.     

Distribution of GABA-like immunoreactive neurons in the optic lobes of Periplaneta americana

Summary Specific antisera against protein-conjugated -aminobutyric acid (GABA) were used in immunocytochemical staining procedures to study the distribution of the putative GABA-like immunoreactive neurons in the optic lobes of Periplaneta. GABA-like immunoreactive structures are evident in all three optic neuropil regions. Six different populations of GABAergic neurons, whose perikarya are grouped around the medulla, are found within the optic lobe. The number of these immunoreactive cells varies greatly and corresponds to the number of ommatidia of the eye. In the proximal part of the lamina, a coarse network of GABA-positive fibres is recognizable. These are the processes of large field tangential cells whose fibres pass through the distal surface of the medulla. A second fibre population of the lamina is made up of the processes of the centrifugal columnar neurons whose perikarya lie proximally to the medulla. The medulla contains 9 layers with GABAergic elements of variable immunoreactivity. Layers 1, 3, 5, 7 and 9 exhibit strong labelling, as a result of partial overlapping of the processes of centrifugal and centripetal columnar neurons, tangential fibres and/or lateral processes of perpendicular fibres and (possibly) processes of amacrines. A strong immunoreactivity is found in the proximal and distal layers of the lobula.     

Distribution of serotonin-like immunoreactive neurons in the slug<Emphasis Type="Italic"> Limax valentianus</Emphasis>

Immunohistochemical techniques were used to study the distribution of serotonin-containing neurons in the nervous system of the slug Limax valentianus. Approximately 350 serotonin-like immunoreactive cell bodies were found in the central nervous system. These were located in the cerebral, pedal, visceral and right parietal ganglia. Most serotonin-like immunoreactive neurons had small cell bodies, which were aggregated into discrete clusters. A pair of previously identified metacerebral giant cells were found on the anterior side of the cerebral ganglion, and two additional pairs of uniquely identifiable, serotonin-like immunoreactive cells were found on the posterior side of the cerebral ganglion. The whole-mount maps of these stained neurons will be useful in further physiological and biochemical studies of olfactory learning at the cellular level in Limax valentianus.This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology, Japan (nos. 12307053 and 13771353)     

Distribution of biogenic amines in the slug,Limax maximus

Summary The distribution of monoamines inLimax maximus was studied by the histochemical fluorescent method of Falck and Hillarp. The number of 5-HT-containing and catecholamine-containing perikarya in the central nervous system is small compared with the non-fluorescent perikarya. However, all the ganglia except the proto-cerebral ganglia have some amine-containing neurons. There are relatively larger numbers of fluorescent cells in the cerebral, visceral, pedal and right parietal ganglia than in the other ganglia. A single, giant 5-HT-containing neuron was observed in each meta-cerebral ganglion.Monoamine neurons are localised in a number of peripheral tissues (heart, integument, tentacles, penis retractor muscle, sole of foot, kidney, alimentary canal, reproductive organs and tentacular, pharyngeal and cephalic retractor muscles). Neurons containing catecholamine are mostly associated with sensory structures such as the statocysts, the retina of the eye and the integument of the tentacles, whereas 5-HT-containing nerve fibres are mainly observed in muscle tissues.We wish to thank the Wellcome Trust for financial support.     

The distribution of GABA-like immunoreactivity in the thoracic nervous system of the locust Schistocerca gregaria

Summary An antiserum raised against gamma aminobuyric acid (GABA) was used to stain the thoracic nervous system of the locust. It stained both neuronal somata and processes within the neuropile. Among the stained somata, those of the three pairs of common inhibitory motor neurones could be identified in each of the three thoracic ganglia. In the pro- and mesothoracic ganglia five discrete groups of somata are stained, four ventral and one dorsal. In the metathoracic neuromere, an additional second dorsal group can be identified. In the abdominal neuromeres of the metathoracic ganglion both dorsal and ventral somata are stained but the latter cannot be divided into discrete populations. In each ganglion, dorsal commissures (DC) IV and V are composed of stained neurites, DCVII, the supramedian commissure, the perpendicular tract, and all the longitudinal tracts contain both stained and unstained neurites. DCI, II, III and VI, the T and I tracts are unstained. An abundance of GABA-like immunoreactive processes is found throughout the neuropile except for the anterior ventral association centre where stained processes are sparser. Some of the stained cell groups contain neurones that have been studied physiologically. The function of these neurones is discussed.Beit Memorial Fellow     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10^-3, 10^-4, 10^-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.     

GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria

Summary Neurones in the suboesophageal ganglion of the locust Schistocerca gregaria were stained with an antiserum raised against gamma amino butyric acid (GABA). This ganglion consists of the fused mandibular, maxillary and labial neuromeres. Immunoreactive cell bodies of similar size and distribution occur in the lateral, ventral and middorsal regions of all three neuromeres. Approximately 200 cell bodies stain in both the mandibular and maxillary neuromeres and 270 in the labial neuromere. A few distinctly larger cells occur in the ventral groups and one large pair occurs in the lateral group of the maxillary neuromere. Dorsal commissures DCIV and DCV are composed mainly of stained fibres, while DCI–DCIII are largely unstained. A ventral commissure also stains in the maxillary neuromere. All longitudinal tracts contain both stained and unstained fibres. Many processes within the neuropil are also immunoreactive. A stained axon is found in the posterior tritocerebral commissure which enters the anterior dorsal region of the mandibular neuromere. The salivary branch of the 7th nerve contains one stained axon and two axons stain in nerve 8 which innervates neck muscles.     

The development of GABA-like immunoreactivity in the thoracic ganglia of the locust Schistocerca gregaria

Summary The development of GABA-like immunoreactivity was investigated in embryonic and juvenile locusts using an antibody raised against GABA-protein conjugates. GABA-like immunoreactivity was first detectable in the neuropile of embryonic ganglia at 55% development, and in neuronal somata at 62% development. The total number of immunoreactive somata increased between 62% and 85% embryonic development, and followed an anterio-posterior pattern of expression. At 85% development, the number of immunoreactive somata reached adult levels and no change in number was then seen. In embryonic stages and first and second juvenile instars two dorsal and four ventral groups of somata were labeled in all three thoracic ganglia, whilst in later juvenile instars one of the dorsal groups was visible as a separate entity only in the metathoracic ganglion. These early patterns were modified by alterations in the positions of some of the groups during late embryogenesis and during juvenile development to produce the adult pattern. The results show that the development of GABA expression is similar to that of other neurotransmitters. The characteristics of the development of immunoreactivity indicate that some of these immunoreactive clusters may be derived from clonally related neurones. Finally, we demonstrate the presence of immunoreactive somata and processes in embryos, which correspond to those of identified local and intersegmental interneurones studied in the adult.Abbreviations Ab1–3 first-third abdominal ganglion - CON connective - CI _1–3 common inhibitors 1–3 - CTC tract - DC I–VII dorsal commissures I–VII - DIT dorsal intermediate tract - DMT dorsal median tract - LDT lateral dorsal tract - LF lateral fibres - o, iLVT outer and inner lateral ventral tract - MVT median ventral tract - N1–5 nerves 1–5 - aPT anterior perpendicular tract - PT perpendicular tract - aRT anterior ring tract - R1–5 nerve roots 1–5 - PVC posterior ventral commissure - SMC supra-median commissure - T3 metathoracic neuromere - TT T tract - aVAC anterior ventral association centre - VC I ventral commissure I - d,vVCII dorsal and ventral parts of ventral commissure II - VF ventral fibres - VIT ventral intermediate tract - VLT ventral lateral tract - VMT ventral median tract - (d,v)LAG (dorsal and ventral) lateral anterior group - LDG lateral dorsal group - LVG lateral ventral group - MDG medial dorsal group - MPG medial posterior group - MVG medial ventral group     

Distribution of GABA-immunoreactive neurons in the forebrain of the goldfish,Carassius auratus

Summary The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the forebrain (tel-and diencephalon) of the goldfish by means of immunocytochemistry on Vibratome sections using antibodies against GABA. Positive perikarya were detected in the olfactory bulbs and in all divisions of the telencephalon, the highest density being found along the midline. In the diencephalon, GABA-containing cell bodies were found in the hypothalamus, in particular in the preoptic and tuberal regions. The inferior lobes, the nucleus recessus lateralis, and more laterodorsal regions, such as the nucleus glomerulosus and surrounding structures, also exhibited numerous GABA-positive perikarya. Cell bodies were also noted in the thalamus, in particular in the dorsomedial, dorsolateral and ventromedial nuclei. The relative density of immunoreactive fibers was evaluated for each brain nucleus and classified into five categories. This ubiquitous distribution indicates that, as in higher vertebrates, GABA most probably represents one of the major neurotransmitters in the brain of teleosts.     

GABA-like immunoreactivity of identified interneurons in the flight system of the locust,Locusta migratoria

Summary The transmitter content of identified inhibitory interneurons in the flight system of the locust, Locusta migratoria, has been characterized using antibodies raised against protein-conjugated gamma aminobutyric acid. Identified flight neurons were filled with the fluorescent dye, Lucifer Yellow. Serial sections of dye-filled neurons were incubated with an antibody to gamma aminobutyric acid which was subsequently tagged with a fluorescent marker. Excitatory motoneurons to wing muscles and 13 flight interneurons (3 excitatory, 7 inhibitory, and 3 with unknown synaptic effect) were examined. Neither the moto-neurons nor any of the 3 excitatory interneurons contained immunoreactive material. Six of the 7 inhibitory interneurons did contain immunoreactive material. All the neurons which contained immunoreactive material and whose synaptic effect is known were inhibitory. We conclude that most of the inhibitory flight interneurons which have been described use gamma aminobutyric acid as their transmitter. Interestingly, at least 1 set of interneurons known to be inhibitory does not use gamma aminobutyric acid. We predict that the 2 interneurons which do contain immunoreactive material and whose synaptic effect is not yet known will be found to have inhibitory roles in the operation of the flight circuitry.     

Cerebral interneurons controlling fictive feeding in Limax maximus

Summary Initiation and modulation of fictive feeding by cerebral to buccal interneurons (CBs) was examined in an isolated CNS preparation of Limax maximus. Three CBs which are phasically active during fictive feeding, CB₁, CB₃ and CB₄, will reliably trigger bouts of fictive feeding when activated alone or in pairs. Another phasic CB, CB_EC, is not effective for triggering feeding. One CB which is tonically active during fictive feeding, CB_ST, drives fictive feeding in 50% of preparations when activated alone and enhances triggering of feeding when co-activated with phasic CBs. The metacerebral giant cell (MGC) was found to be capable of triggering fictive feeding in preparations with an intact subcerebral commissure. The MGC was especially effective at increasing the effectiveness of other CBs for initiation of feeding. Short high-frequency bursts of phasic CB or MGC action potentials are capable of resetting ongoing fictive feeding. Resetting effects of CB action potentials are relatively independent of the phase of the bite-cycle in which they are activated. CB₄ phase-advances the bite-cycle while the other phasic CBs phase-delay the bite cycle. Moderate frequency stimulation of CB₄ speeds up the bite rate while moderate frequency stimulation of CB₃ slows biting. All CBs, except the tonic CB, CB_DL, increase the intensity of buccal motor neuron bursting during feeding. The excitatory effects of phasic CBs and the tonic CB, CB_EPSP, on fictive feeding persist for many seconds after the offset of stimulation. CBs form both monosynaptic excitatory and monosynaptic inhibitory connections with different BG motor neurons.Abbreviations BG buccal ganglion - BR buccal root - CB cerebral-buccal interneuron - CBC cerebral-buccal connective - CPG central pattern generator - FB fast burster neuron - FMP feeding motor program - IBI interbite interval - MGC metacerebral giant cell     

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

Distribution of calbindinlike immunoreactive structures in the optic tectum of normal and eye-enucleated cyprinid fish

Summary Using the ABC immunohistochemical method, we investigated the distribution of calbindinlike immunoreactive structures in the optic tectum of normal fish, Tinca tinca, and from normal and unilaterally eye-enucleated fish, Cyprinus carpio. In nonoperated individuals of both species the optic tectum contained numerous immunoreactive neurons with strongly positive somata located in the stratum periventriculare and a thick immunolabeled dendritic shaft ascending radially toward the stratum fibrosum et griseum superficiale. The retinorecipient layers contained many fibrous immunoreactive structures. Some varicose fibers, isolated or in small bundles, were localized to the stratum album centrale, especially in the dorsal tectal half. Unilateral eye removal produced the disappearance of the immunoreactive fibrous structures located in the retinorecipient layers of the tectum contralateral to the enucleation. The present work shows that calbindinlike immunoreactive substances are localized in specific neural circuits of the fish optic tectum and suggests that the calbindin-like immunoreactive fibers in the retinorecipient strata are of retinal origin.     

Immunohistochemical demonstration of cholinergic structures in central ganglia of the slug (Limax maximus, Limax valentianus)

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans.     

Distribution of GABA-like immunoreactivity during post-metamorphic development and regeneration of the central nervous system in the ascidian Ciona intestinalis

During regeneration of the neural ganglion in Ciona intestinalis, the pattern of reappearance of some peptidergic cells is similar to the ontogenetic patterns exhibited by these cell types during normal post-metamorphic development. Using a specific antiserum to gamma-aminobutyric acid (GABA), we describe here the appearance of GABA-ergic cells in Ciona during both post-metamorphic development and regeneration of the neural ganglion following total ablation. Post-metamorphic animals were divided into the categories: 1, 3–5, 6–10, 11–15 and 23–27 mm in body length. Regeneration was monitored at 12, 15, 18, 21, 28 and 56 days post ablation. The first appearance of GABA-like immunoreactive cells during normal development were at the 3 to 5-mm stage where they were seen as discrete cells, without processes, evenly distributed in the cortical region throughout the ganglion. Fibres were first seen at the 6 to 10-mm stage. As development proceeded, GABA-like immunoreactive cells became more concentrated near the nerve root exits and along the dorsal rind of the ganglion. In regenerating ganglia, GABA was first detected at 18–21 days post ablation, in cells that lacked any obvious processes and that were distributed in all regions of the ganglion. At 28 days post ablation, processes could be detected in the neuropile, and after 56 days GABA cells were found predominantly in the same regions as in the normally developing adult ganglion. Although the overall pattern reflects that in a normal adult, a few differences were detectable. For example, rather more GABAergic cells were concentrated ventrally in the ganglion close to the neural gland.     

Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis

Summary Development of the nervous system of the pluteus larva of Strongylocentrotus droebachiensis was investigated using indirect immunofluorescence with antibodies against dopamine, GABA, and serotonin, and glyoxylic acid-induced fluorescence of catecholamines. Serotonergic cells first appear in full gastrulae; dopaminergic and GABAergic cells are present in early four-arm plutei. The number of neurons and the complexity of the nervous system increases through development of the pluteus. In the pluteus the dopaminergic component of the nervous system includes a ganglion in the lower lip of the mouth and a pair of ganglia at the base of the post-oral arms which extend axons along the base of the circumoral ciliary band. The distribution of cells visualized by glyoxylic acid-induced fluorescence is similar to that of dopaminergic cells. GABAergic neurons occur in the upper lip and in the wall of the esophagus. Serotonergic neurons are present in the lower lip; the pre-oral hood contains an apical ganglion which extends axons along the base of the epidermis overlying the blastocoel. The dopaminergic and GABAergic components of the nervous system are associated with effectors involved in feeding and swimming. The serotonergic component is not associated with any apparent effectors but may have a role in metamorphosis.     

Repeatability of energy metabolism and resistance to dehydration in the invasive slug Limax maximus

Standard metabolic rate (SMR) and resistance to body dehydration (BD) are important physiological traits that have an effect on water balance and the amount of energy available for activity and production, and thus could contribute to variation in life history traits expressed across a range of environments. Few studies have tested whether SMR and BD show consistent between‐individual variation in molluscs. Significant repeatability of SMR and BD indicates that the traits might be heritable and therefore a possible target for natural selection, so describing the repeatability of SMR and BD is important in studies of phenotypic variability. Here, we studied energy metabolism (body mass‐corrected SMR) and the change in the scaling relationship of SMR and body mass in response to time between measurements in the giant garden slug Limax maximus. Limax maximus is one of the most invasive terrestrial molluscs, with a wide geographical distribution, and is considered an important pest of horticultural and agricultural crops. Our results show that L. maximus follows the expected relationship of increasing SMR with increasing mass, but the scaling exponent varies through time and is different from that described for other gastropods. We also found significant inter‐individual variation in VCO_{2 Mean}, VCO_{2 Min}, VCO_{2 Max}, and BD (τ=0.25, 0.29, 0.24, 0.22, p<0.05, respectively), and significant repeatability of body mass (τ=0.90). To our knowledge, this is the first comprehensive analysis of the repeatability of body mass‐corrected SMR and BD in terrestrial slugs. Our results suggest that energy metabolism and water balance could potentially respond to selection.