Accurate automated quantitative imaging of tortoise erythrocytes using the nis image analysis system

Abstract

The standard method for assessing blood cell characteristics using an ocular micrometer is time-consuming and limited. We used the Nikon NIS Elements imaging software and May- Grünwald-Giemsa staining to determine whether automated image analysis is suitable for rapid and accurate quantitative morphometry of erythrocytes. Blood was collected during four seasons from 126 geometric tortoises and the blood smears were evaluated for cell (C) and nuclear (N) characteristics of the erythrocytes. We measured area, length (L), width (W), perimeter, elongation and pixelation intensity, and calculated L/W and N/C areas.

Erythrocyte size differed among cohorts; females, the larger sex, had smaller erythrocytes than either males or juveniles. Males had more elongated erythrocytes than females and erythrocytes of adults were more elongated than those of juveniles. Erythrocyte size and shape influence the efficiency of gas exchange owing to surface area to volume ratios, which are greater for small, elongated cells than for large, round cells. The high N/C ratio and low pixelation intensities of males and juveniles indicate that they may have had more immature erythrocytes in their circulation than females. The use of pixelation intensity to indicate the presence of immature erythrocytes was validated by seasonal differences that corresponded to the biology of the tortoises. Pixelation intensity was lowest in winter. We found that automated image analysis is a rapid and reliable method for determining cell size and shape, and it offers the potential for distinguishing among developmental stages that differ in staining intensity. The method should be useful for rapid health assessments, particularly of threatened species, and for comparative studies among different vertebrates.     

Body size development of captive and free‐ranging Leopard tortoises (Geochelone pardalis)

The growth and weight development of Leopard tortoise hatchings (Geochelone pardalis) kept at the Al Wabra Wildlife Preservation (AWWP), Qatar, was observed for more than four years, and compared to data in literature for free‐ranging animals on body weight or carapace measurements. The results document a distinctively faster growth in the captive animals. Indications for the same phenomenon in other tortoise species (Galapagos giant tortoises, G. nigra; Spur‐thighed tortoises, Testudo graeca; Desert tortoises, Gopherus agassizi) were found in the literature. The cause of the high growth rate most likely is the constant provision with highly digestible food of low fiber content. Increased growth rates are suspected to have negative consequences such as obesity, high mortality, gastrointestinal illnesses, renal diseases, “pyramiding,” fibrous osteodystrophy or metabolic bone disease. The apparently widespread occurrence of high growth rates in intensively managed tortoises underlines how easily ectothermic animals can be oversupplemented with nutrients. Zoo Biol 29:517–525, 2010. © 2009 Wiley‐Liss, Inc.     

Are tortoises important seed dispersers in Amazonian forests?

According to most studies on seed dispersal in tropical forests, mammals and birds are considered the main dispersal agents and the role played by other animal groups remains poorly explored. We investigate qualitative and quantitative components of the role played by the tortoise Chelonoidis denticulata in seed dispersal in southeastern Amazon, and the influence of seasonal variation in tortoise movement patterns on resulting seed shadows. Seed shadows produced by this tortoise were estimated by combining information on seed passage times through their digestive tract, which varied from 3 to 17 days, with a robust dataset on movements obtained from 18 adult C. denticulata monitored with radio transmitters and spoon-and-line tracking devices. A total of 4,206 seeds were found in 94 collected feces, belonging to 50 seed morphotypes of, at least, 25 plant genera. Very low rates of damage to the external structure of the ingested seeds were observed. Additionally, results of germination trials suggested that passage of seeds through C. denticulata’s digestive tract does not seem to negatively affect seed germination. The estimated seed shadows are likely to contribute significantly to the dispersal of seeds away from parent plants. During the dry season seeds were dispersed, on average, 174.1 m away from the location of fruit ingestion; during the rainy season, this mean dispersal distance increased to 276.7 m. Our results suggest that C. denticulata plays an important role in seed dispersal in Amazonian forests and highlight the influence of seasonal changes in movements on the resulting seed shadows.     

Isolation and characterization of eight microsatellite loci for the study of gene flow between Testudo marginata and Testudo weissingeri (Testudines: Testudinidae)

The taxonomic status of the Greek Testudo species, marginated tortoise (Testudo marginata Schoepff, 1793) and dwarf marginated tortoise (Testudo weissingeri Bour, 1995), is controversed. In order to study the gene flow among these Greek Testudo species, eight polymorphic microsatellite markers were characterized and screened for 32 individuals. The number of alleles observed per locus ranged from two to 19. Observed heterozygosities varied from 0.235 to 0.830. These microsatellite loci were also successfully tested on seven closely related species of Testudinidae. This set of microsatellites offers an efficient tool to investigate genetic differentiation among testudinid species.     

Thermal variability in body temperature in an ectotherm: Are cloacal temperatures good indicators of tortoise body temperature?

Historically, studies of reptilian thermal biology have compared ambient temperatures (T_a) to body temperatures (T_b) from the animal under study, with T_b usually taken from the cloaca and various instruments being used to measure T_b. The advent of surgically implanted miniature temperature loggers has offered the opportunity to test the efficacy of cloacal T_b as a measurement in thermoregulatory studies. We expected that there was a difference between skin, cloacal, and core T_b's. Temperatures were measured from various positions on leopard tortoises (Stigmochelys pardalis) using thermocouples and miniature temperature loggers, including surgically implanted temperature loggers. Measurements of temperature from various positions on and in the tortoise were significantly different from T_a. Cloacal T_b's were significantly lower than all other body temperatures measured, and core T_b's were significantly different from cloacal T_b, skin and carapace temperatures. In addition, significant differences were found between measures of temperature from other parts of the body. The variations between core T_b, cloacal T_b and other measures of T_b indicated that there are large thermal gradients within the body of a relatively large tortoise at any given time with cloacal T_b not an accurate measure of core T_b.     

Rewilding the tropics,and other conservation translocations strategies in the tropical Asia‐Pacific region

Alarm over the prospects for survival of species in a rapidly changing world has encouraged discussion of translocation conservation strategies that move beyond the focus of ‘at‐risk’ species. These approaches consider larger spatial and temporal scales than customary, with the aim of recreating functioning ecosystems through a combination of large‐scale ecological restoration and species introductions. The term ‘rewilding’ has come to apply to this large‐scale ecosystem restoration program. While reintroductions of species within their historical ranges have become standard conservation tools, introductions within known paleontological ranges—but outside historical ranges—are more controversial, as is the use of taxon substitutions for extinct species. Here, we consider possible conservation translocations for nine large‐bodied taxa in tropical Asia‐Pacific. We consider the entire spectrum of conservation translocation strategies as defined by the IUCN in addition to rewilding. The taxa considered are spread across diverse taxonomic and ecological spectra and all are listed as ‘endangered’ or ‘critically endangered’ by the IUCN in our region of study. They all have a written and fossil record that is sufficient to assess past changes in range, as well as ecological and environmental preferences, and the reasons for their decline, and they have all suffered massive range restrictions since the late Pleistocene. General principles, problems, and benefits of translocation strategies are reviewed as case studies. These allowed us to develop a conservation translocation matrix, with taxa scored for risk, benefit, and feasibility. Comparisons between taxa across this matrix indicated that orangutans, tapirs, Tasmanian devils, and perhaps tortoises are the most viable taxa for translocations. However, overall the case studies revealed a need for more data and research for all taxa, and their ecological and environmental needs. Rewilding the Asian‐Pacific tropics remains a controversial conservation strategy, and would be difficult in what is largely a highly fragmented area geographically.     

Changes in the Alyki Kitrous wetland in northern Greece: 1990–1999, and future prospects

The Alyki wetland in northern Greece is a site of notable herpetological and ornithological importance. A large population of the tortoise Testudo hermanni began to recover slowly (r = 0.016) between 1990 and 1999, following catastrophic habitat destruction in 1980 and slow decline from 1980–1990. Heathland vegetation showed signs of desiccation, probably due to increased drainage by the salt works, including deterioration of Crataegus, Ruscus and Asphodelus, and loss of formerly important food plants of tortoises. Activity and body mass condition of tortoises were reduced in summer in grassy heath habitats. Predation of artificial (chicken egg) tortoise nests increased, and the relative frequency of juveniles (about 1 % of samples) was lower than after severe habitat destruction or pesticide damage. These changes were attributed to an increasing population of badgers, Meles meles. Population levels of waders were lower in 1998/1999 than in 1988/1989, especially avocets (Recurvirostra avosetta) for which the site was internationally important. Breeding populations of most waders and larids also declined. Mediterranean gulls, for which Alyki was formerly the most important breeding area outside Russia, did not nest from 1990 to 1998, and nests were unsuccessful in 1999. Changes in breeding populations were due to falling lagoon levels, exposure to predators and vegetation encroachment on islet breeding sites. Midwinter waterfowl populations remained high or increased, however, as alternative feeding areas were available in the lagoon. Preservation of the habitats against development of the salt works and increasing desiccation are necessary to maintain the long-term conservation value of the Alyki site.     

The evolution of different maternal investment strategies in two closely related desert vertebrates

We compared egg size phenotypes and tested several predictions from the optimal egg size (OES) and bet‐hedging theories in two North American desert‐dwelling sister tortoise taxa, Gopherus agassizii and G. morafkai, that inhabit different climate spaces: relatively unpredictable and more predictable climate spaces, respectively. Observed patterns in both species differed from the predictions of OES in several ways. Mean egg size increased with maternal body size in both species. Mean egg size was inversely related to clutch order in G. agassizii, a strategy more consistent with the within‐generation hypothesis arising out of bet‐hedging theory or a constraint in egg investment due to resource availability, and contrary to theories of density dependence, which posit that increasing hatchling competition from later season clutches should drive selection for larger eggs. We provide empirical evidence that one species, G. agassizii, employs a bet‐hedging strategy that is a combination of two different bet‐hedging hypotheses. Additionally, we found some evidence for G. morafkai employing a conservative bet‐hedging strategy. (e.g., lack of intra‐ and interclutch variation in egg size relative to body size). Our novel adaptive hypothesis suggests the possibility that natural selection favors smaller offspring in late‐season clutches because they experience a more benign environment or less energetically challenging environmental conditions (i.e., winter) than early clutch progeny, that emerge under harsher and more energetically challenging environmental conditions (i.e., summer). We also discuss alternative hypotheses of sexually antagonistic selection, which arise from the trade‐offs of son versus daughter production that might have different optima depending on clutch order and variation in temperature‐dependent sex determination (TSD) among clutches. Resolution of these hypotheses will require long‐term data on fitness of sons versus daughters as a function of incubation environment, data as yet unavailable for any species with TSD.     

Implications of Anthropogenic Landscape Change on Inter-Population Movements of the Desert Tortoise (Gopherus agassizii)

In the Sonoran Desert of North America, populations of the desert tortoise (Gopherus agassizii) occur in rocky foothills throughout southwestern Arizona and northwestern Mexico. Although tortoise populations appear to be isolated from each other by low desert valleys, individuals occasionally move long distances between populations. Increasingly, these movements are hindered by habitat fragmentation due to anthropogenic landscape changes. We used molecular techniques and radiotelemetry to examine movement patterns of desert tortoises in southern Arizona. We collected blood samples from 170 individuals in nine mountain ranges and analyzed variability in seven microsatellite loci to determine genetic differentiation among populations. Gene flow estimates between populations indicate that populations exchanged individuals historically at a rate greater than one migrant per generation, and positive correlation between genetic and geographic distance of population pairs suggests that the limiting factor for gene flow among populations is isolation by distance. Life history traits of the desert tortoise, a long-lived species with delayed sexual maturity, may severely constrain the ability of small populations to respond to disturbances that increase adult mortality. Historic gene flow estimates among populations suggests that recovery of declining populations may rely heavily on the immigration of new individuals from adjacent mountain ranges. Management strategies compatible with the evolutionary history of gene flow among disjunct populations will help ensure the long-term persistence of Sonoran desert tortoise populations.