MHC class I of saltwater crocodiles (Crocodylus porosus): polymorphism and balancing selection

Saltwater crocodiles are in high demand for the production of luxury fashion items. However, their susceptibility to disease incurs substantial losses and it is hoped to be able to genetically select these animals for disease resistance. So far, this has only been enabled by phenotypic selection. Investigating the major histocompatibility complex (MHC) could provide insight into the ability of an individual to respond to pathogens acting as a selective pressure on the host. Here, we assessed genetic diversity and a role of selection in shaping the diversity of MHC class I exon 3 among 42 saltwater crocodiles from nine river basins in the Northern Territory, Australia. We generated 640 sequences using cloning and sequencing methods and identified 43 MHC variants among them. Phylogenetic analyses clustered these variants into two major clades, which may suggest two gene lineages. We found the number of variants within an individual varying between one and seven, indicating that there are at least four gene loci in this species. Selection detection analyses revealed an elevated ratio of nonsynonymous to synonymous substitutions (mean?=?1.152 per codon), suggesting balancing selection. Population differentiation analyses revealed that the MHC did not show structuring among the river basins, and there were some shared variants among them. This may be a result of possible gene flow and/or similar selection pressures among populations. These findings provide background knowledge to identify potential MHC markers, which could be used for selecting genetically variable individuals for future disease associations. All MHC class I exon 3 sequences reported in this paper were submitted to the GenBank database with following accession numbers: HQ008785–HQ008789, HQ008791–HQ008798, HQ008808–HQ008815, HQ008824, HQ008826–HQ008830, HQ008835, HQ008839, HQ008842–HQ008850, and JX023536–JX023540.     

Morphometric analysis of embryonic development in Alligator mississippiensis, Crocodylus johnstoni and Crocodylus porosus

The size of embryos at various stages of development was determined in three species of crocodilian ( Alligator mississippiensis, Crocodylus johnstoni and C. porosus). Various morphometric measurements were taken of embryos throughout development and were described for each stage of development. Increase in size from stage to stage was faster in A. mississippiensis than in C. porosus and C. johnstoni. Hatchlings of A. mississippiensis were large in length but light in mass compared with the hatchlings of C. johnstoni and C. porosus which were heavier per unit length. These morphometric parameters can be used to determine the stage of embryonic development by size. The use of principle component analysis improves this technique further by dampening any anomalous data points.
The rate of embryonic growth in A. mississippiensis appeared to be under greater genetic control than in the two species of Crocodylus. The evolutionary advantages of this phenomenon probably relate to the biology of A. mississippiensis. Due to the northerly range of this species it is advantageous for alligators to hatch as soon as possible, as large as possible, to maximize the period prior to winter hibernation and reduce predation. Tropical crocodiles have fewer selection pressures for rapid development and have slower rates of embryonic growth. Genetic aspects of crocodilian embryonic development have been largely ignored but may help explain some aspects of crocodilian growth under farming conditions.     

253 Novel polymorphic microsatellites for the saltwater crocodile (Crocodylus porosus)

Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253 novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an average polymorphic information content (PIC) of 0.425.     

Exercise training enhances aerobic capacity in juvenile estuarine crocodiles (Crocodylus porosus)

Aerobic capacity (VO2max) of endothermic vertebrates is known to increase with exercise training, but this effect has not been found to-date in non-avian reptiles. We exercised juvenile estuarine crocodiles (Crocodylus porosus) to walk at 0.75-0.88 km/h on a treadmill for up to 20 min a day over 16 weeks, and compared their aerobic performance with that of unexercised crocodiles. In the exercised group, VO2max increased from 6.9 to 8.5 mLO2/kg/min (+28%), and locomotor endurance increased from 3.8 to 6.9 min (+82%). Neither VO2max nor endurance changed significantly in the sedentary group. This finding extends the exercise training effect onto another vertebrate clade, and demonstrates that ectothermic amniotes are capable of elevating their aerobic capacity in response to exercise training. We propose that differences in cardiopulmonary structure and function in non-avian reptiles may be responsible for the absence (in squamates) or presence (in crocodilians) of a strong training effect on aerobic capacity.     

湾鳄(Crocodylus porosus)线粒体基因组全序列结构分析与鳄类系统发生

该文测序了湾鳄的线粒体基因组全序列,全长为16,917bp。湾鳄mtDNA结构与其他脊椎动物相似,由22个tRNA,2个rRNA和13个蛋白质编码基因及1个非编码的控制区(D-loop)所组成。除NADH6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(UCN)、tRNAGlu、tRNAPro在L-链上编码之外,其余基因均在H-链编码。基因排列顺序与已测序的鳄类一致,这显示了鳄类线粒体基因排列顺序上的保守性。但鳄类线粒体基因排列顺序与脊椎动物的典型排列方式相比,有较大的差异,尤其是tRNAPhe基因的重排、tRNASer-tRNAHis-tRNALeu基因族的排列方式等。湾鳄mtDNA和已测序的鳄类一样,缺失轻链复制起始点(OLR)。基于17种鳄mtDNA控制区保守区,采用PAUP4.0最大简约法(Maximumparsimony,MP)构建MP树,邻接法(Neighbor-joiningmethod,NJ)构建NJ树,结果显示:食鱼鳄(Gavialisgangeticus)和假食鱼鳄(Tomistomaschlegelii)聚为一支后再与鳄科(Crocodylidae)的其他物种形成姐妹群,这与基于食鱼鳄和假食鱼鳄的线粒体全序列的分析结果一致,支持将食鱼鳄并入鳄科的观点。结果还支持非洲窄吻鳄(Crocodyluscataphractus)与鳄属(Crocodylus)构成姐妹群,可以单独划分为属的观点。     

QTL mapping for two commercial traits in farmed saltwater crocodiles (Crocodylus porosus)

The recent generation of a genetic linkage map for the saltwater crocodile (Crocodylus porosus) has now made it possible to carry out the systematic searches necessary for the identification of quantitative trait loci (QTL) affecting traits of economic, as well as evolutionary, importance in crocodilians. In this study, we conducted genome‐wide scans for two commercially important traits, inventory head length (which is highly correlated with growth rate) and number of scale rows (SR, a skin quality trait), for the existence of QTL in a commercial population of saltwater crocodiles at Darwin Crocodile Farm, Northern Territory, Australia. To account for the uncommonly large difference in sex‐specific recombination rates apparent in the saltwater crocodile, a duel mapping strategy was employed. This strategy employed a sib‐pair analysis to take advantage of our full‐sib pedigree structure, together with a half‐sib analysis to account for, and take advantage of, the large difference in sex‐specific recombination frequencies. Using these approaches, two putative QTL regions were identified for SR on linkage group 1 (LG1) at 36 cM, and on LG12 at 0 cM. The QTL identified in this investigation represent the first for a crocodilian and indeed for any non‐avian member of the Class Reptilia. Mapping of QTL is an important first step towards the identification of genes and causal mutations for commercially important traits and the development of selection tools for implementation in crocodile breeding programmes for the industry.     

Morphology and ultrastructure of possible integumentary sense organs in the estuarine crocodile (Crocodylus porosus)

The skins of crocodylids and gavialids can be distinguished from those of alligatorids by the presence of darkly pigmented pits, known as integumentary sense organs (ISOs), on the postcranial scales. The structure of ISOs, in Crocodylus porosus, was studied using light microscopy and scanning and transmission electron microscopy. The stratum corneum of the epidermis in the area of the ISO is thinner, while the stratum germinativum is thicker, relative to other regions of the integument. Beneath the epidermal layer the ISO region has a paucity of collagen fibers relative to the rest of the dermis. Widely dispersed fibrocytes, nerve terminals, and chromatophores occur throughout the ISO region of the dermis, but these elements are concentrated in the area immediately beneath the stratum germinativum in the ISO region. The morphology of the ISOs suggests that they are sensory organs. It has traditionally been assumed that sensory organs on the amniote integument have a mechanosensory function. However, alternate functional interpretations of this structure are possible, and a resolution awaits further work. © 1996 Wiley-Liss, Inc.     

Scaling of standard metabolic rate in estuarine crocodiles Crocodylus porosus

Standard metabolic rate (SMR, ml O₂ min^?1) of captive Crocodylus porosus at 30 °C scales with body mass (kg) according to the equation, SMR = 1.01 M^0.829, in animals ranging in body mass of 3.3 orders of magnitude (0.19–389 kg). The exponent is significantly higher than 0.75, so does not conform to quarter-power scaling theory, but rather is likely an emergent property with no single explanation. SMR at 1 kg body mass is similar to the literature for C. porosus and for alligators. The high exponent is not related to feeding, growth, or obesity of captive animals. The log-transformed data appear slightly curved, mainly because SMR is somewhat low in many of the largest animals (291–389 kg). A 3-parameter model is scarcely different from the linear one, but reveals a declining exponent between 0.862 and 0.798. A non-linear model on arithmetic axes overestimates SMR in 70 % of the smallest animals and does not satisfactorily represent the data.     

Differences in renal-cloacal function between Crocodylus porosus and Alligator mississippiensis have implications for crocodilian evolution

Major electrolytes and nitrogenous excretory products were analysed in the blood plasma, ureteral urine and cloacal urine of juvenile Alligator mississippiensis and Crocodylus porosus in fresh and hypoosmotic salt water (206 mosmol · l⁻¹). Both species coped well with saline water, showing little (Alligator) or no (Crocodylus) change in plasma composition. Comparisons of renal-cloacal function point to major differences in their osmoregulatory physiology. The cloaca of C. porosus is a very active osmoregulatory organ in salt and fresh water, contributing to water conservation and NaCl excretion through the lingual salt glands. In contrast, the cloaca of Alligator has little impact on the composition of excreted urine. It seems likely that A.␣mississippiensis is largely constrained to a renal response to osmotic and ionic stress while C. porosus is able to call on a more complex mix of renal response, post-renal modification of urine in the cloaca, and excretion of excess NaCl through the salt glands. The results support the idea that there are deep-seated differences in the osmoregulatory physiology of alligatorids and crocodylids (Eusuchia), an understanding of which should provide valuable insights into their evolution and zoogeography. Accepted: 7 September 1996     

Survival and growth of hatchling Crocodylus porosus in saltwater without access to fresh drinking water

Summary It has been suggested that C. porosus select nest sites which provide a source of freshwater for hatchlings during the dry season. From a mark-recapture study, we conclude that hatchling C. porosus can survive and grow in hyperosmotic saltwater without drinking freshwater. Hence, the siting of nests is unlikely to be the consequence of a requirement by hatchlings for freshwater. Considered along with other information, our observations imply that hatchling C. porosus have functional salt glands.     

Nitric oxide synthase in the gastrointestinal tract of the estuarine crocodile, Crocodylus porosus

The aim of this study was to investigate the distribution of nitric oxide synthase (NOS)-containing nerve cells in the gastrointestinal tract of a reptile and to compare it with the pattern in other vertebrate classes. In the estuarine crocodile, Crocodylus porosus, NOS-positive nerve cell bodies and fibres were found in all regions of the gut examined. Most myenteric microganglia contained one or several NOS-immunoreactive neurons together with unlabelled neurons. The majority of the neurons were multipolar, ranging from 10 to 25 microns in diameter. Both the circular and the longitudinal muscle layers were innervated by NOS-immunoreactive nerve fibres, which mostly ran parallel to the muscle fibres. In addition, small blood vessels in the submucosa and on the serosal surface of the gut were innervated by NOS-immunoreactive fibres. Double labelling with antisera to NOS and vasoactive intestinal peptide (VIP) revealed three neuronal subpopulations. A small proportion of the NOS-immunoreactive cells also contained immunoreactivity to VIP while a majority of the VIP-immunoreactive cells were NOS immunoreactive. There were more nerve fibres showing VIP immunoreactivity than fibres with NOS immunoreactivity, although most of the latter also contained immunoreactivity to VIP. VIP-immunoreactive fibres often surrounded the NOS-immunoreactive nerve cells. These results suggest that neuronally released nitric oxide is likely to be involved in the control of gastrointestinal motility in the crocodile as in most other vertebrate species.     

Deurithitrema gingae n.g., n.sp. (Digenea: Plagiorchioidea) from the kidneys of a salt-water crocodile (Crocodylus porosus Schneider) from Australia

Summary The name Deurithitrema gingae n. g., n. sp. is proposed for worms from the kidneys of one Crocodylus porosus from Darwin, Northern Territory, Australia. The following combination of characters distinguishes the new genus from all others: prepharynx very short; oesophagus absent; caeca reaching to posterior end of body; testes tandem in middle third of body, contiguous or amost so; cirrus-sac large, over-reaches ventral sucker, contains bipartite seminal vesicle, prostatic regions and ejaculatory duct with spines around its opening into genital atrium; genital pore immediately in front of ventral sucker; uterus coiled to posterior end of body; vitellaria lateral from ventral sucker to slightly behind testes; excretory vesicle small, receiving a single ventro-lateral duct from each side behind end of uterine loops. Deurithitrema belongs to the superfamily Plagiorchioidea Dollfus, 1930. It superficially resembles a number of genera, but its relationships within the superfamily are obscure.     

The calibre of the Foramen of Panizza in Crocodylus porosus is variable and under adrenergic control

The foramen of Panizza is located within the outflow tract of the crocodilian heart, between the left and right aortas. It has been suggested that the foramen of Panizza has a variable calibre, which could explain the profound changes in the distribution of flows and pressure profiles recorded in the right and left aortas. We investigated this possibility using a modified in-situ perfused heart preparation in combination with isolated strip preparations from the outflow tract. In the perfused heart preparation, bolus injections of adrenaline increased the resistance in the foramen of Panizza, indicating a decrease in its diameter. Isolated strip preparations from the outflow tract showed a concentration-dependent increase in tension in response to adrenaline, while vasoactive intestinal polypeptide caused a relaxation in adrenaline pre-contracted strip preparations. We propose that an increase in the diameter of the foramen of Panizza may be important during pulmonary to systemic shunts to allow blood to flow from the left to right aorta (reverse foramen flow) in order to supply the carotid and coronary arteries. During non-shunting conditions, a constricted foramen may prevent excess flow from the right to left aorta during diastole.     

Food capture, appetite, digestion rate and efficiency in hatchling and juvenile Crocodylus porosus

Juvenile estuarine crocodiles captured insects and crabs at or above the water surface by leaps and lunges powered by the hind limbs and tail. The mouth opened as the head cleared the water; most prey were held by sidesnaps of the jaws. Such prey capture was accurate, deliberate and preceded by stalking. In contrast, submerged prey (e.g. prawns) appeared to be detected mainly by touch and detection was followed by undirected, inaccurate 'snapping frenzies' which were usually ineffective. Small prey items were swallowed whole under water. Large dense prey (e.g. crabs) were handled and swallowed on land or in very shallow water; large less dense prey (e.g. cockroaches) were swallowed during vigorous water-treading in deep water, the head being maintained above the water surface.
Young crocodiles ate satiation meals of 9–10% body weight (on a fresh food weight basis) at 30°C, and appetite returned over about 40 h as the stomach emptied. Total gut clearance time for a meal was 4–5 d. Evidence was obtained which demonstrated that young Crocodylus porosus Schneider deliberately eat gastroliths from an early stage. Such gastroliths are retained within the stomach (as were barium/polystyrene spheroids of 1 mm diameter) presumably by the action of the well-developed pyloric sphincter. X-radiographs demonstrated that gastroliths are dispersed throughout the stomach contents after a meal and presumably aid digestion.
Assimilation rates for dry mass (77.5%), energy (85.2%) and protein-N (97.4%) were high in normal juveniles. Animals exhibiting 'runt syndrome' showed strong appetite but slow food processing by the gut, together with poor assimilation, especially of protein (35.7%).     

Comparison of stress induced by manual restraint and immobilisation in the estuarine crocodile,Crocodylus porosus

This study compared the stress induced in captive estuarine crocodiles, Crocodylus porosus, by two different handling methods: manual restraint (noosing with ropes) and immobilization by electro-stunning. To stun, a short charge (approx. 6 s) at 110 V was delivered to the back of the necks of C. porosus using a custom-built device, which immobilized the animals for 5-10 min. Immobilized and restrained animals were measured and sexed, and the condition of the skin assessed. Blood samples were taken from some animals immediately after restraint or immobilization. Other animals were returned to their pens to recover for periods of 30 min, 1, 4, 12, 24 or 48 hours after which they were stunned and blood samples taken. Individual animals (mean body length 1.96 m, N=99) were bled only once. Haematocrit and haemoglobin concentrations were measured and plasma samples were analysed for corticosterone, glucose and lactate levels. Following restraint, there were significant increases in haematocrit, haemoglobin, glucose, lactate and corticosterone concentrations in C. porosus. For restrained animals, recovery to baseline levels occurred after approximately 8 hours. The stress response of stunned animals was significantly reduced compared to manually captured and restrained crocodiles. Both groups showed a significant increase in haematocrit, haemoglobin concentration and lactate levels, however the magnitude of change was significantly reduced, and recovery was faster in stunned animals. No increase in either glucose or corticosterone levels occurred with immobilisation. The results imply that immobilization by electro-stunning is much less stressful.     

Control of the development of the pulmonary surfactant system in the saltwater crocodile,Crocodylus porosus

Pulmonary surfactant is a mixture of lipids and proteins that controls the surface tension of the fluid lining the inner lung. Its composition is conserved among the vertebrates. Here we hypothesize that the in ovo administration of glucocorticoids and thyroid hormones during late incubation will accelerate surfactant development in the saltwater crocodile, Crocodylus porosus. We also hypothesize that the increased maturation of the type II cells in response to hormone pretreatment will result in enhanced responsiveness of the cells to surfactant secretagogues. We sampled embryos at days 60, 68, and 75 of incubation and after hatching. We administered dexamethasone (Dex), 3,5,3'-triiodothyronine (T(3)), or a combination of both hormones (Dex + T(3)), 48 and 24 h before each prehatching time point. Lavage analysis indicated that the maturation of the phospholipids (PL) in the lungs of embryonic crocodiles occurs rapidly. Only T(3) and Dex + T(3) increased total PL in lavage at embryonic day 60, but Dex, T(3), and Dex + T(3) increased PL at day 75. The saturation of the PLs was increased by T(3) and Dex + T(3) at day 68. Swimming exercise did not increase the amount or alter the saturation of the surfactant PLs. Pretreatment of embryos with Dex, T(3), or Dex + T(3) changed the secretion profiles of the isolated type II cells. Dex + T(3) increased the response of the cells to agonists at days 60 and 68. Therefore, glucocorticoids and thyroid hormones regulate surfactant maturation in the crocodile.     

Morphology of the cloaca in the estuarine crocodile, Crocodylus porosus, and its plastic response to salinity

The cloacal complex of Crocodylus porosus is composed of three chambers (proctodaeum, urodaeum, and coprodaeum) separated by tight, muscular sphincters. The proctodaeum is proximal to the cloacal vent and houses the genitalia. The urodaeum is the largest chamber, is capable of storing large quantities of urine, and is lined with an epithelium with the capacity for transepithelial water and ion exchange. The coprodaeum, the most orad cloacal chamber, is a small, only marginally expandable chamber that has an epithelium composed almost entirely of mucus-secreting cells. The coprodaeum and lower intestine are reported to be the site(s) for urine modification in birds and bladderless lizards. A radiographic trace of urine storage in C. porosus kept for 2 months under hyperosmotic conditions showed no signs of retrograde movement of urine into the coprodaeum or rectum. Instead, urine was stored in the urodaeum of C. porosus. Examination of the mucosal surface of the urodaeum by SEM showed a plastic response to environmental salinity, with a possible increase in surface area in animals kept in hyperosmotic water compared with animals from fresh water. We propose the urodaeum as the primary site for postrenal modification of urine in C. porosus.