Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.     

Developmental morphology of the neonatal alligator (Alligator mississippiensis) ovary

American alligator (Alligator mississippiensis) ovary development is incomplete at hatching. During the months following hatching, the cortical processes of oogenesis started in ovo continues and folliculogenesis is initiated. Additionally, the medullary region of the gonad undergoes dramatic restructuring. We describe alligator ovarian histology at hatching, 1 week, 1 month, and 3 months of age in order to characterize the timing of morphological development and compare these findings to chicken ovary development. At hatching, the ovarian cortex presents a germinal epithelium containing oogonia and a few primary oocytes irregularly scattered between somatic epithelial cells. The hatchling medulla shows fragmentation indicative of the formation of lacunae. By 1 week of age, oocytes form growing nests and show increased interactions with somatic cells, indicative of the initiation of folliculogenesis. Medullary lacunae increase in diameter and contain secretory material in their lumen. At 1 month, nest sizes and lacunar diameters continue to enlarge. Pachytene oocytes surrounded by somatic cells are more frequent. Trabeculae composed of dense irregular connective tissue divide cortical nests. Three months after hatching oocytes in meiotic stages of prophase I up to diplotene are present. The ovary displays many enlarged follicles with oocytes in diplotene arrest, thecal layers, lampbrush chromosomes, and complete layers of follicular cells. The medulla is an elaborated complex of vascularized lacunae underlying the cortex and often containing discrete lymphoid aggregates. While the general morphology of the alligator ovary is similar to that of the chicken ovary, the progression of oogenesis and folliculogenesis around hatching is notably slower in alligators. Diplotene oocytes are observed at hatching in chickens, but not until 3 months in alligators. Folliculogenesis is completed at 3 weeks in chickens whereas it is still progressing at 3 months in alligators.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

Production of superoxide ions by leukocytes of the American alligator (Alligator mississippiensis)

This study was conducted to characterize the production of superoxide ions by leukocytes in whole blood of the American alligator (Alligator mississippiensis). We used WST-1, a tetrazolium salt which can be reduced to a water-soluble formazan compound with high molar absorptivity at 438 nm, to probe the production of superoxide by alligator leukocytes. Incubation of alligator whole blood with WST-1 resulted in a time- and concentration-dependent increase in absorbance of the plasma at 438 nm. The reduction of WST-1 was inhibited in a concentration-dependent manner by superoxide dismutase, an enzyme that catalyzes the reduction of superoxide to peroxide, confirming that the reduction of WST-1 was due to the presence of superoxide. Treatment of whole blood with nitrotetrazolium blue (NBT) resulted in the staining of heterophils and monocytes, enforcing the idea that that the production of superoxide is due to the presence of leukocytes, and not other blood cell components. It is interesting to note that the production of superoxide by the alligator leukocytes required no external stimulation while human leukocytes must be stimulated with an immunological challenge before producing superoxide. This is the first report of the production of superoxide as an innate immune mechanism in crocodilians.     

Antibacterial properties of serum from the American alligator (Alligator mississippiensis)

Treatment of alligator (Alligator mississippiensis) and human serum samples with Escherichia coli resulted in a time- and concentration-dependent inhibition of bacterial proliferation. When inoculated with E. coli, alligator serum exhibited 10-fold lower bacterial survival rates after 1 h than human serum. In addition, the antibacterial spectrum of alligator serum was shown to be much broader than that of human serum, with growth inhibition occurring in 100% of bacterial strains tested (compared to only 35% for human serum). Additional results showed that the antimicrobial activities of alligator serum could be completely inhibited by preincubation with proteases, indicating the proteinaceous nature of the antimicrobial activities. Furthermore, incubation of alligator serum at 56 degrees C for 30 min (classical human serum complement inactivation conditions) obliterated all antimicrobial properties of the alligator serum. The antibacterial activities occurred relatively quickly in vitro, with significant activity occurring within 5 min of inoculation with E. coli and maximal activity at 20 min. Also, the antimicrobial activity exhibited temperature dependence, with a substantial decrease in activity below 15 degrees C. These data suggest that the antimicrobial properties of alligator serum may be due to an active serum complement system.     

Amoebacidal effects of serum from the American alligator (Alligator mississippiensis)

Treatment of axenic Naegleria gruberi cultures with alligator serum resulted in time-dependent amoebacidal activity, with measurable activity at 5 min and maximal activity occurring at 20 min. The amoebacidal activity was concentration dependent, with measurable activity at 25% serum, whereas treatment of amoebas with undiluted serum resulted in only 16% survival. The efficacy was dependent on the concentration of amoebas, with higher survival rates at high amoeba densities and lower survival rates at low amoeba densities. The amoeba-killing effects of alligator serum were broad in spectrum because the serum was effective against 3 strains of Naegleria species tested and 4 Acanthamoeba species, which have been reported to be resistant to human serum complement-mediated lysis. The amoebacidal effects of alligator serum were temperature dependent, with optimal activity at 15-30 C and a decrease in activity below 15 C and above 30 C. The amoebacidal activity of alligator serum was heat labile and protease sensitive, indicating the proteinaceous nature of the activity, and was also inhibited by ethylenediaminetetraacetic acid, which indicated a requirement for divalent metal ions. These characteristics strongly suggest that the amoebacidal properties of alligator serum are because of complement activity.     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Isolation of faecal coliform bacteria from the American alligator (Alligator mississippiensis)

Aims: To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina’s coastal waters. Methods and Results: Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most‐probable‐number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8·0 × 10⁹ g^?1 (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. Conclusions: A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. Significance and Impact of the Study: These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.     

Febrile response to infection in the American alligator (Alligator mississippiensis)

Temperature probes were inserted into the stomachs of juvenile American alligators (Alligator mississippiensis) maintained outdoors at ambient fluctuating temperatures. Internal body temperatures (T_b) were measured every 15 min for two days, and then the alligators were injected with bacterial lipopolysaccharide (LPS), pyrogen-free saline, or left untreated. Alligators injected intraperitoneally with LPS exhibited maximum T_bs 2.6 ± 1.1 °C and 3.5 ± 1.2 °C higher than untreated control animals on days one and two after treatment, respectively. T_bs for these animals fell to within control ranges by day three postinjection. Similarly, mean preferred body temperatures (MPBTs) were significantly higher for LPS-injected alligators on days one (4.2 ± 1.8 °C) and two (3.5 ± 1.6 °C) after treatment. Intraperitoneal injection of heat-killed Aeromonas hydrophila, a gram-negative bacterium known to infect crocodilians, resulted in a fever while injection of Staphylococcus aureus (gram positive) did not elicit a febrile response. Injection of LPS in alligators maintained indoors in a constant temperature environment resulted in no increase in internal T_b. These results indicate that alligators did not exhibit a febrile response in the absence of a thermal gradient, and suggest that febrile responses observed are probably behavioral in nature.     

On the median pharyngeal valve of the American alligator (Alligator mississippiensis)

The middle ear cavities of crocodilians have complex connections with the pharyngeal lumen, including lateral and median components which both open into a single chamber located on the dorsal midline of the pharynx. This chamber and the surrounding soft-tissue is herein termed the median pharyngeal valve. In the American alligator (Alligator mississippiensis) this valve opens, for a duration of 0.3 s, approximately every 120 s; the patency of the median pharyngeal valve was not influenced by either auditory stimuli or by submersing the alligator underwater. The median pharyngeal valve has an outer capsule of dense connective tissue and fibrocartilage and an inner “plug” of loose connective tissue. These opposing surfaces are lined by respiratory epithelium and separated by a cavity that is continuous with the middle ear cavities and the pharyngeal lumen (through a central opening in the capsule termed the pore). The inner plug of the median pharyngeal valve is contacted by skeletal muscles positioned to serve as both elevators/retractors (which would open the valve) and elevators/protractors (which, in conjunction with gravity, would close the valve). Unlike other vertebrate valve systems, the median pharyngeal valve appears to function as a deformable ball check valve.     

Isolation of polymorphic microsatellite loci from the Chinese alligator (Alligator sinensis)

Ten microsatellite loci were isolated from the Chinese alligator (Alligator sinensis), using a partial genomic DNA library and an enrichment method. Fifty microsatellite repeats were screened from the library of 888 positive clones, 10 of which were polymorphic. These microsatellite loci developed here are another set of novel molecular markers for A. sinensis, and they will be suitable for population genetic studies as well as for kinship analysis.     

Hind limb movements of the American alligator (Alligator mississippiensis) and postural grades

Middle ear ontogeny in Monodelphis domestica is investigated to understand better both the immediate consequences for suckling in a neonate marsupial and the epigenetic factors that constrain morphogenesis. Neonates of Monodelphis possess neither mammalian (dentarysquamosal) nor reptilian (quadrate-articular) jaw articulations, nor does the contact between the incus and crista parotica offer a joint surface ( contra Maier, 1987). Elasticity in Meckel's cartilage allows minimal deflection of the lower jaw. Observation of the developmental rate of the individual elements reveals that mandibular arch derivatives (malleus, incus and tensor tympani) are on a slightly faster ontogenetic schedule than hyoid arch derivatives (stapes and m. stapedius).     

Histochemical study of jaw muscle fibers in the American alligator (Alligator mississippiensis)

The ontogenetic development of caudal vertebrae and associated skeletal elements of salmonids provides information about sequence of ossification and origin of bones that can be considered as a model for other teleosts. The ossification of elements forming the caudal skeleton follows the same sequence, independent of size and age at first appearance. Dermal bones like principal caudal rays ossify earlier than chondral bones; among dermal bones, the middle principal caudal rays ossify before the ventral and dorsal ones. Among chondral bones, the ventral hypural 1 and parhypural ossify first, followed by hypural 2 and by the ventral spine of preural centrum 2. The ossification of the dorsal chondral elements starts later than that of ventral ones. Three elements participate in the formation of a caudal vertebra: paired basidorsal and basiventral arcocentra, chordacentrum, and autocentrum; appearance of cartilaginous arcocentra precedes that of the mineralized basiventral chordacentrum, and that of the perichordal ossification of the autocentrum. Each ural centrum is mainly formed by arcocentral and chordacentrum. The autocentrum is irregularly present or absent. Some ural centra are formed only by a chordacentrum. This pattern of vertebral formation characterizes basal teleosts and primitive extant teleosts such as elopomorphs, osteoglossomorphs, and salmonids. The diural caudal skeleton is redefined as having two independent ural chordacentra plus their arcocentra, or two ural chordacentra plus their autocentra and arococentra, or only two ural chordacentra. A polyural caudal skeleton is identified by more than two ural centra, variably formed as given for the diural condition. The two ural centra of primitive teleosts may result from early fusion of ural centra 1 and 2 and of ural centra 3 and 4, or 3, 4, and 5 (e.g., elopomorphs), respectively. The two centra may corespond to ural centrum 2 and 4 only (e.g., salmonids). Additionally, ural centra 1 and 3 may be lost during the evolution of teleosts. Additional ural centra form late in ontogeny in advanced salmonids, resulting in a secondary polyural caudal skeleton. The hypural, which is a haemal spine of a ural centrum, results by growth and ossification of a single basiventral ural arococentrum and its haemal spine. The proximal part of the hypural always includes part of the ventral ural arcocentrum. The uroneural is a modification of a ural neural arch, which is demonstrated by a cartilaginous precursor. The stegural of salmonids and esocids originates from only one paired cartilaginous dorsal arcocentrum that grows anteriorly by a perichondral basal ossification and an anterodorsal membranous ossification. The true epurals of teleosts are detached neural spines of preural and ural neural arches as shown by developmental series; they are homologous to the neural spines of anterior vertebrae. Free epurals without any indication of connection with the dorsal arococentra are considered herein as an advanced state of the epural. Caudal distal radials originate from the cartilaginous distal portion of neural and haemal spines of preural and ural (epurals and hypurals) vertebrae. Therefore, they result from distal growth of the cartilaginous spines and hypurals. Cartilaginous plates that support rays are the result of modifications of the plates of connective tissue at the posterior end of hypurals (e.g., between hypurals 2 and 3 in salmonids) and first preural haemal spines, or from the distal growth of cartilaginous spines (e.g., epural plates in Thymallus). Among salmonids, conditions of the caudal skeleton such as the progressive loss of cartilaginous portions of the arcocentra, the progressive fusion between the perichondral ossification of arcocentra and autocentra, the broadening of the neural spines, the enlargement and interdigitation of the stegural, and other features provide evidence that Prosopium and Thymallus are the most primitive, and that Oncorhynchus and Salmo are the most advanced salmonids respectively. This interpretation supports the current hypothesis of phylogenetic relationships of salmonids. © 1992 Wiley-Liss, Inc.     

Formation and regression of the corpus luteum of the American alligator (Alligator mississippiensis)

Luteal morphology of the American alligator is unique when compared to other reptiles but is similar to that of its phylogenetic relatives, the birds. The theca is extensively hypertrophied, but the granulosa never fills the cavity formed following the ovulation of the ovum. The formation of the corpus luteum (CL) is correlated with elevated plasma progesterone concentrations, which decline dramatically after oviposition with the onset of luteolysis. Unlike those of most other reptiles, the central luteal cell mass is composed of two cell types; one presumably is derived from the granulosa, whereas the other is from the theca interna. Both cell types are present throughout gravidity but only one cell type is seen during mid to late luteolysis. A significant decline in luteal volume occurs following oviposition and continues throughout the post-oviposition period. The fastest decline in luteal volume occurs in the month immediately after oviposition; this rate then slows. Luteolysis appears to continue for a year or more following oviposition, as distinct structures of luteal origin can still be identified in animals 9 months after oviposition. The size of persistent CL can be used to determine whether a given female oviposited during the previous nesting season. Females with CL having volumes greater than 0.2 cm² or CL diameters greater than 0.4 cm were active the previous season. © 1995 Wiley-Liss, Inc.     

Developmental effects of embryonic exposure to toxaphene in the American alligator (Alligator mississippiensis)

A variety of organochlorine pesticides have been shown to adversely affect embryonic development. A number of abnormalities have been documented in alligators (Alligator mississippiensis) from highly-contaminated Lake Apopka, FL, USA that are similar to the results of experimental studies exposing embryos to pesticides. In the current study, we exposed developing alligator embryos to varying concentrations of toxaphene, a broad-spectrum pesticide found in relatively high concentration in Lake Apopka alligator egg yolk. The toxaphene, dissolved in 50 microl of ethanol, was applied topically to the eggshell just prior to the sex-determining period of development. Shortly after hatching, we examined a number of morphological and physiological endpoints to determine the consequences of sub-lethal embryonic exposure to toxaphene. Our results indicate that toxaphene had little or no effect on the morphological endpoints examined including body mass (BM) and size, liver, thyroid, and gonad development. In addition, toxaphene failed to affect sexual differentiation, or in vitro thyroxin, testosterone (T), and estradiol production. However, male plasma T concentration was higher in animals treated with 10 and 0.01 microg toxaphene/kg (based on mean egg mass) than control males. Because in vitro T production was not different among control groups, we suggest the difference in plasma T could be due to differences in hypothalamic-pituitary stimulation of the gonad or hepatic steroid degradation. This study indicates that technical grade toxaphene, at the applied doses, does not induce the same developmental abnormalities associated with alligators living in Lake Apopka. Future studies should consider the effects of embryonic exposure to a mixture of chemicals, including toxaphene metabolites, on development in alligators to better evaluate the consequences of environmental contamination.     

Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry

There are many computer programs that can match tandem mass spectra of peptides to database-derived sequences; however, situations can arise where mass spectral data cannot be correlated with any database sequence. In such cases, sequences can be automatically deduced de novo, without recourse to sequence databases, and the resulting peptide sequences can be used to perform homologous nonexact searches of sequence databases. This article describes details on how to implement both a de novo sequencing program called “Lutefisk,” and a version of FASTA that has been modified to account for sequence ambiguities inherent in tandem mass spectrometry data.     

野生扬子鳄种群及栖息地现状研究

1999年7～8月及2000年8～9月,利用GPS、激光测距仪等,采用夜间灯光照射计数方法,对 有野生扬子鳄(Alligator sinensis)存在的安徽省宣州、泾县、广德、郎溪、南陵等5 县市的26个地点进行了调查,包括扬子鳄国家级自然保护区的13个指定保护点。结果发现： 目前野生扬子鳄主要生存在第一类栖息地（1999年50.7%、2000年40.0%）,面积为17.38 hm²;其他两类栖息地的野生扬子鳄分布比率较小（各为1999年24.0%、2000年30.9%、1999 年25.3% 、2000年29.1%）,面积分别为22.04hm²、19.03hm²。两年的平均生态密度分别为1.28条/hm²和1.79条/hm²,野生扬子鳄种群数量为145条。其种群已明显分为至少18个数 量不等且相互隔离的地方种群。建议恢复足够大的栖息地,并放饲养鳄于其中以重新 建立有效野生种群。     

Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligator mississippiensis)

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brains of the American alligator, Alligator mississippiensis, using reverse-phase high-pressure liquid chromatography (HPLC). The concentration of total GnRH was 8.8 ng/g of frozen brain tissue or 21.1 ng per brain. The amino acid sequence of each form of GnRH was determined using automated Edman degradation. The presence of the N-terminal pGlu residue was established by digestion studies with bovine pyroglutamyl aminopeptidase and coelution with synthetic forms of the native peptide. The primary structure of alligator GnRH I is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 and alligator GnRH II is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.