Mercury in Morelet's crocodile eggs from northern Belize.

Recent studies have examined mercury accumulation in crocodilians. However, though most researchers have focused on tissue concentrations, few have examined mercury levels in crocodilian eggs. In July 1995, we analyzed mercury in 31 nonviable Morelet's crocodile ( Crocodylus moreletii) eggs collected from eight nests across three localities in northern Belize. All eggs were found to contain mercury. Based on an individual egg basis, mean concentration of mercury for all three localities was among the lowest reported for any crocodilian species. When localities were examined separately, mean concentrations for Laguna Seca and Gold Button Lagoon were comparable to those observed in other studies, and the mean for Sapote Lagoon was the lowest ever reported. Based on mean nest concentrations, mercury in eggs from Laguna Seca was approximately two- and tenfold higher than for Gold Button Lagoon and Sapote Lagoon, respectively. Variability in mercury concentrations among localities is likely the result of site-specific differences in mercury input, bioavailabilty, and bioaccumulation. Mercury concentrations were relatively uniform in eggs from the same nest and among nests from the same localities. The presence of mercury in Morelet's crocodile eggs suggests exposure in adult females, developing embryos, and neonates. However, crocodiles in these areas show no overt signs of mercury toxicity, and no indication of population decline is evident. A paucity of data on the effects of mercury on crocodilians precludes meaningful speculation as to the biological significance of tissue and egg concentrations. Controlled laboratory studies and long-term population monitoring are needed to address these questions.

Ticks from a Morelet's crocodile in Belize.

Parasitism of crocodilians by ticks has rarely been reported, and to our knowledge only seven published accounts exist. On 3 July 1999, we collected four ticks from a subadult Morelet's crocodile (Crocodylus moreletii) captured in northern Belize. These were identified as Amblyomma dissimile (one female), and Amblyomma sp. (two nymphs, one larva). The crocodile was captured on land approximately 100 m from water, and all four ticks were attached to loose skin on the lateral surface of the tail. Crocodilians are most susceptible to terrestrial ectoparasites, including ticks, during overland movements. However, most such movements occur in response to drought, when tick questing activity is suppressed, which likely accounts for the small numbers of tick specimens recorded from crocodilians and the absence of any noticeable impact of parasitism on host fitness.

Falcaustra kutcheri n. sP. (nematoda: kathlaniidae) from Geoemyda yuwonoi (testudines: emydidae) from Sulawesi, Indonesia.

Falcaustra kutcheri n. sp. (Nematoda: Kathlaniidae) from feces of the turtle Geoemyda yuwonoi is described and illustrated. Falcaustra kutcheri n. sp. represents the 68th species to be assigned to this genus and is distinguished from other oriental species by the distribution pattern of caudal papillae of the male: 10 pairs of sessile caudal papillae, 4 pairs precloacal, 1 pair adcloacal, 5 pairs postcloacal.

Organochlorine contaminants in Morelet's crocodile (Crocodylus moreletii) eggs from Belize.

Non-viable eggs of Morelet's crocodile (Crocodylus moreletii) were collected from Gold Button (GBL) and New River lagoons (NRL) in northern Belize and screened for organochlorine (OC) compounds using gas chromatography (GC) with electron capture detection (ECD). All egg samples from both lagoons (n = 24) tested positive for one or more OCs. Primary contaminants were p,p-DDE and methoxychlor, detected in 100% and 29% of the eggs examined, respectively. Concentrations of individual OC contaminants ranged from 1 ppb (ng chemical/g egg) to > 0.5 ppm (microgram chemical/g egg). Total concentrations of OCs (sum of all OCs) for one egg collected from a nest at GBL reached as high as 0.7 ppm. Sediment samples from both lagoons also tested positive for OCs (lindane, aldrin, methoxychlor, heptachlor epoxide, p,p-DDT, among others). Nest media (soil and plant material) collected from crocodile nests at GBL were positive for p,p-DDT, methoxychlor, aldrin, endosulfan II, and endrin aldehyde. Based on the 24 egg samples analyzed to date, crocodiles from both lagoons are being exposed to OCs. Such exposure may present a health threat to populations of crocodiles in Central America.

Organochlorine contaminants in Morelet's crocodile (Crocodylus moreletii) eggs from Belize

Non-viable eggs of Morelet's crocodile (Crocodylus moreletti) were collected from Gold Botton (GBL) and New River lagoons (NRL) in northern Belize and screened for organochlorine (OC) compounds using gas chromatography (GC) with electron capture detection (ECD). All egg samples from both lagoons (n=24) tested positive for one or more OCs. Primary contaminants were p,p-DDE and methoxychlor, detected in 100 percent and 29 percent of the eggs examined, respectively. Concentrations of individual OC contaminants ranged from 1 ppb (ng chemical/g egg). Total concentrations of OCs (sum of all OCs) for one egg collected from a nest at GBL reached as high as 0.7 ppm. Sediment samples from both lagoons also tested positive for OCs (lindane, aldrin, methoxychlor, heptachlor epoxide, p, p-DDT, among others). Nest media (soil and plant material) collected from crocodile nests at GBL were positive for p, p-DDT, methoxychlor, aldrin, endosulfan II, and endrin aldehyde. Based on the 24 egg samples analyzed to date, crocodiles from both lagoons are being exposed to OCs. Such exposure may present a health threat to populations of crocodiles in Central America. (AU)

DDE in eggs of two crocodile species from Belize.

Organochlorine (OC) residues were recently detected in nonviable Morelet's crocodile (Crocodylus moreletii) eggs from northern Belize. To further the assessment of contaminant exposure in Belizean crocodiles, nonviable Morelet's crocodile eggs (n = 11) from southern Belize and American crocodile (Crocodylus acutus) eggs (n = 12) from the coastal zones of Belize were screened for 20 OCs. Results indicated p,p-DDE to be the most prevalent OC (96% occurrence) in eggs examined, with concentrations ranging from 5 to 372 ng/g. These concentrations are similar to those observed in crocodile eggs (10-180 ng/g) from northern Belize. A general trend toward higher DDE concentrations in Morelet's crocodile eggs (mean = 103 ppb) compared with American crocodile eggs (mean = 31 ppb) was observed. However, this trend may be due to site-specific contamination rather than differences in interspecific susceptibility to chemical exposure. Other OCs detected in crocodile eggs included the parent compound, p,p-DDT, and its metabolite, p, p-DDD.

Ectromelia in Morelet's crocodile from Belize.

Two Morelet's crocodiles (Crocodylus moreletii) captured on 21 March 1997 and 20 April 1998 in the New River system, Belize exhibited ectromelia of one forelimb. External and radiograph examination appears to indicate limb agenesis of unknown etiology, as there is no apparent scarring or skeletal trauma. These two individuals represent the only cases of missing limbs from 642 individuals captured in this study and to our knowledge, the first reported cases in Morelet's crocodile. Several factors including age and diet of the reproducing female, extremes in nest conditions (egg incubation temperature and humidity), and exposure to environmental contaminants can cause developmental abnormalities in crocodilians and may have contributed to the condition observed in these animals. Survival rates for hatchling crocodilians are generally low, and embryonic malformations such as ectromelia may constitute an added disadvantage to survival. However, both individuals examined in this study were vigorous and appeared in good condition.

Ectromelia in Morelet's crocodile from Belize

Two Morelet's crocodiles (Crocodylus moreletti) captured on 21 March 1997 and 20 April 1998 in the New River system, Belize exhibited ectromelia of one forelimb. External and radiograph examination appears to indicate limb agenesis of unknown etiology, as there is no apparent scarring or skeletal trauma. These two individuals represent the only cases of missing limbs from 642 individuals captured in this study and to our knowledge, the first reported cases in Morelet's crocodile. Several factors including age and diet of the reproducing female, extremes in nest conditions (egg incubation temperature and humidity), and exposure to environmental contaminants can cause developmental abnormalities in crocodilians and may have contributed to the condition observed in these animals. Survival rates for hatching crocodilians are generally low, and embryonic malformations such as ectromelia may constitute an added disadvantage to survival. However, both individuals examined in this study were vigorous and appeared in good condition.(Au)

Dot assay for neomycin phosphotransferase activity in crude cell extracts.

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.

Detection of opines by colorimetric assay.

A colorimetric procedure for confirming the presence of arginine-derived opines (nopaline and octopine) in plant tissue extracts is described. Those materials are widely used as markers of plant cell transformation and tumorigenesis mediated by the tumor-inducing plasmids of Agrobacterium tumefaciens. Nopaline and octopine are generally detected, following resolution by paper electrophoresis, by observation of the uv-fluorescent products formed upon reaction with phenanthrenequinone. We found that a further heat treatment step, compatible with paper electrophoresis, results in rapid production of a red-purple pigment. Our colorimetric assay is sensitive to 1.25-micrograms quantities of opine and eliminates problems of background fluorescence encountered with crude plant extract in the usual assay.

Nucleotide sequence of the gene encoding the matrix protein of Newcastle disease virus.

The nucleotide sequence of the gene encoding the matrix (M) protein of the Beaudette C strain of Newcastle disease virus (NDV) has been determined from overlapping cDNA clones. Control sequences typical of paramyxovirus mRNA start and polyadenylation signals have been identified. Assuming that the M gene starts and finishes at these sequences, the M gene is 1241 nucleotides long and encodes one long open reading frame of 364 amino acids, corresponding to a polypeptide of molecular weight 39605, in good agreement with estimates from SDS gels. The M protein has an amino acid sequence that is both hydrophobic and highly basic. The NDV M protein has sequence homologies to the M proteins of Sendai, measles, canine distemper and respiratory syncytial viruses.

Opine synthesis in wild-type plant tissue.

Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid.

Ammonia accumulation and inhibition of photosynthesis in methionine sulfoximine treated spinach.

Ammonia accumulation and photosynthetic rate inhibition took place when spinach leaf tissue was supplied with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. This effect was observed in the absence of significant inorganic nitrogen reduction or an exogenous source of ammonia. Both the time lag prior to the initial photosynthetic rate decrease and the rate of that decrease depend on the O(2) and MSO concentrations supplied to the leaf tissue. However, the total rate of ammonia accumulation was similar at both 20% and 2.2% O(2). The decline in photosynthetic rate was not caused by stomatal closure but may be a result of ammonia toxicity. The data point out the importance of glutamine synthetase in preventing the poisoning of leaf metabolism by ammonia generated internally through processes not involved in net nitrogen assimilation. The rapidity of the action of MSO in suppressing photosynthesis was unexpected and should not be overlooked in interpreting data from other experiments involving that inhibitor. MSO shows promise as a tool for investigating C-N flow, particularly during photorespiration.

Photosynthesis and increased production of protein.

Photosynthesis, the use of light energy in the conversion of CO2 and inorganic nutrients into plant material, is the ultimate source of the food protein necessary to man's existence. Given certain assumptions, the overall maximal theoretical photosynthetic efficiency of agricultural plants can be calculated. Actual measured maximal growth rates of plants are equivalent to efficiency levels well below that theoretical maximum. In air, C4 plants can some closer to the theoretical value than C3 plants, perhaps because C4 plants avoid the occurrence of measurable photorespiration and oxygen inhibition of photosynthesis. Alfalfa, a C3 legume, is an extremely productive protein source. Its protein yield per acre can surpass that of commonly grown C4 crops (corn, sorghum) and C3 seed crops (soybean, wheat, rice). Alfalfa leaf protein is of high nutritional quality and can apparently be used directly in the human diet, eliminating the protein loss involved in animal production. Plant protein productivity can be raised as part of an increase in overall crop yield. The growth of plants in atmospheres with elevated CO2 levels can result in increased yields. In C3 plants this is due, at least in part, to the suppression of photorespiration and oxygen inhibition of photosynthesis. We have investigated the effect of CO2 concentration on alfalfa photosynthetic metabolism. Our results support the contention that alfalfa productivity can be increased by an environment of elevated CO2. A second approach toward increased plant protein productivity is through regulation of carbon flow during photosynthesis so as to increase portein production relative to that of other plant constituents. In particular, we have investigated whether ammonia (the form in which plants first incorporate nitrogen) can act to regulate leaf carbon metabolism. Our results indicate that NH4+, in part through stimulation of pyruvate kinase, brings about increased production of amino acids at the expense of sucrose production in alfalfa. The effect may be of considerable importance in the regulation of green leaf protein synthesis.

Ammonia regulation of carbon metabolism in photosynthesizing leaf discs.

Alfalfa (Medicago sativa L., var. El Unico) leaf discs, floating on buffer containing NH(4)Cl and photosynthesizing with (14)CO(2), produced more labeled amino acid and less sucrose than did control discs (no added NH(4)Cl). The level of pyruvate increased and that of phosphoenolpyruvate decreased. These and other changes in levels of labeled compounds led us to conclude that pyruvate kinase was activated by ammonia, resulting in increased transfer of photosynthetically incorporated carbon to synthesis of amino acid skeletons at the expense of sucrose synthesis. Carbon flow through enzymes catalyzing the anaplerotic reactions was apparently stimulated.

Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration.

When the CO(2) concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO(2) in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO(2)) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO(2), providing further evidence that the cycle is well regulated.

Analysis of steady state photosynthesis in alfalfa leaves.

A method for carrying out kinetic tracer studies of steady state photosynthesis in whole leaves has been developed. An apparatus that exposes whole leaves to (14)CO(2) under steady state conditions, while allowing individual leaf samples to be removed as a function of time, has been constructed. Labeling data on the incorporation of (14)C into Medicago sativa L. metabolite pools are reported. A carbon dioxide uptake rate of 79 micromoles (14)CO(2) per milligram chlorophyll per hour was observed at a CO(2) level slightly below that of air. Several actively turning over pools of early and intermediate metabolites, including 3-phosphoglyceric acid, glycerate, citrate, and uridine diphosphoglucose, showed label saturation after approximately 10 to 20 minutes of photosynthesis with (14)CO(2) under steady state conditions. Alanine labeling increased more rapidly at first, and then at a lower rate as saturation was approached. Sucrose was a major product of photosynthesis and label saturation of the sucrose pool was not observed. Labeled carbon appeared rapidly in secondary metabolites. The steady state apparatus used has numerous advantages, including leaf temperature control, protection against leaf dehydration, high illumination, known (14)CO(2) specific radioactivity, and provision for control and adjustment of (14)CO(2) concentration. The apparatus allows for experiments of long duration and for sufficient sample points to define clearly the metabolic steady state.