Characterization,multiplex conditions,and cross‐species utility of tetranucleotide microsatellite loci for Blanchard's cricket frog (Acris crepitans blanchardi)

We have isolated and characterized 17 tetranucleotide microsatellite loci for Blanchard's cricket frog (Acris crepitans blanchardi), an anuran common in the central USA. Sixteen loci were organized into four multiplex amplification reactions. These loci were highly polymorphic when screened in 55 individuals from two distant populations, with 11–48 alleles per locus (average = 24.8). Observed and expected heterozygosities ranged from 0.18 to 0.97 and from 0.17 to 0.96, respectively. Nine loci were also polymorphic in Acris crepitans crepitans, with seven polymorphic in Acris gryllus. Five loci amplified in all three taxa. These loci will be useful for population‐ and species‐level investigations of this widespread group.     

Estimated absorbance spectra of the visual pigments of the North Atlantic right whale (Eubalaena glacialis)

To assess the spectral sensitivities of the retinal visual pigments from the North Atlantic right whale (Eubalaena glacialis), we have cloned and sequenced two exons from the rod opsin gene and two exons from the middle‐wavelength sensitive (MWS) cone opsin gene in order to determine the amino acids at positions known to be key regulators of the spectral location of the absorbance maximum (λ_max). Based on previous mutagenesis models we estimate that the right whale possesses a rod visual pigment with a λ_max of 499 nm and a MWS cone visual pigment with a λ_max of 524 nm. Although the MWS cone visual pigment from the right whale is blue‐shifted in its spectral sensitivity like those from odontocetes, the spectral sensitivity of the right whale rod visual pigment is similar to those from terrestrial mammals.     

Simulation of the entanglement of a North Atlantic right whale (Eubalaena glacialis) with fixed fishing gear

Population estimates of the critically endangered North Atlantic right whale (Eubalaena glacialis) put the number of individuals at 458 with the actual number likely being lower due to a recent unusual mortality event. Entanglement with fixed fishing gear is the most significant cause of mortality of North Atlantic right whales. There remains little documentation of how North Atlantic right whales become enwrapped during an encounter with fixed fishing gear. In order to gain a better understanding of how entanglements might occur, an interactive simulator was developed that allows the user to swim a virtual whale model using a standard game controller through a gear field in an attempt to re‐create an entanglement. The morphologically accurate right whale model produces realistic swimming motions and is capable of pectoral fin motions in response to user input. Using the simulator, gear entanglements involving the pectoral flippers including ropes wrapping around the body and entanglements involving the tailstock were re‐created. Entanglements involving the pectoral flippers with body wraps were more easily generated than entanglements involving the tailstock only. The simulator should aid scientists, fisheries experts, fishing gear designers, and bycatch reduction scientists in understanding entanglement dynamics and testing potential new gear configurations.     

Computational fluid dynamics of flow regime and hydrodynamic forces generated by a gliding North Atlantic right whale (Eubalaena glacialis)

Accurate estimates of drag on marine animals are required to investigate the locomotive cost, propulsive efficiency, and the impacts of entanglement if the animal is carrying fishing gear. In this study, we performed computational fluid dynamics analysis of a 10 m (length over all) right whale to obtain baseline measurements of drag on the animal. Swimming speeds covering known right whale speed range (0.125 m/s to 8 m/s) were tested. We found a weak dependence between drag coefficient and Reynolds number. At a swimming speed of 2 m/s, we analyzed the boundary layer thicknesses, the flow regimes, and drag components. We found the thickest boundary layer at the lateral sides of the peduncle, whereas the boundary layer thickness over the outer part of the flukes was less than 1.7 cm. Laminar flow occurred over the anterior ~0.6 LoA and turbulent flow from ~0.8 LoA to the fluke notch. On the surfaces of the flukes outside of the body wake region, flow was laminar. Our most significant finding is that the drag coefficient (0.0071–0.0059) of a right whale for swimming speeds ranging from 0.25 m/s to 2 m/s is approximately twice that of many previous estimates for cetaceans.     

Isolation,characterization and multiplex genotyping of raccoon tetranucleotide microsatellite loci

We have isolated and characterized 12 polymorphic tetranucleotide microsatellite loci in the raccoon (Procyon lotor). Three multiplex panels comprising the 12 loci were developed and 80 individuals from southeastern and western Ontario were genotyped; allele sizes were assigned without difficulty. One locus isolated was identified as an X‐linked marker. The number of alleles per locus ranged from six to 25 with the average heterozygosity ranging from 0.674 to 0.925. These loci will be used to characterize raccoon population structure across North America, and the data used to further understand the spread of raccoon rabies.     

PCR multiplexing of nuclear microsatellite loci inQuercus species

Simple sequence repeats have become the tool of choice in a wide range of studies of forest trees. Despite frequent use of multicolor fluorescent labeling DNA fragment analyzers, there are few procedures that reduce time and cost of the analyses by PCR multiplexing. Here we present an example of 2 multiplexes of 7 unlinked nuclear microsatellite loci to increase the efficiency of genotyping of large samples of oaks, which is extremely useful in population genetic studies.     

Quantification and genetic profiling of DNA isolated from free-floating feces of the North Atlantic right whale (Eubalaena glacialis)

Fecal analysis from the highly endangered North Atlantic right whale provides valuable information about health and reproductive parameters of individual animals. Genetically profiling the feces facilitates this connection when the sample originator is unknown. Although genetic analysis of feces collected in terrestrial systems has become well established, genetic studies of cetacean DNA are rare. Here, the use of free‐floating feces as a source of right whale DNA and the reliability of the genotypes produced are examined. On average, fecal extracts yielded 25 ng of DNA/mg dry weight, but less than 1% was right whale DNA. Although all samples were amplified using genus‐specific mitochondrial control region primers, the quantity of right whale DNA present was over estimated when compared to amplifications using nuclear primers. No correlation was found between the quantity of right whale DNA recovered and the duration the sample sat in the water. Composite microsatellite profiles from multiple amplifications of 28 fecal samples of known origin were consistent with profiles of the same individuals obtained from skin biopsies, however, the rate of allelic dropout varied depending on the amount of right whale DNA added. A screening and genotyping protocol for reliable genetic profiling based on fecal DNA quantification is presented.     

North Pacific right whale (Eubalaena japonica) seasonal and diel calling patterns from long‐term acoustic recordings in the southeastern Bering Sea, 2000–2006

We assessed North Pacific right whale (Eubalaena japonica) seasonal and daily calling patterns in the southeastern Bering Sea (SEBS) using long‐term hydrophone recordings from October 2000 through January 2006. We detected right whale calls on the SEBS middle shelf (<100 m depth) as early as May, intermittently throughout summer and fall, and as late as December. Calls also were detected on one day in June 2005 on the SEBS slope (>1,000 m), but were not detected near Kodiak Island from April to August 2003. In months with calls, detections occurred on more days in July–October (≥6 d/mo), than from May to June or November to December (≤3 d/mo). Calls were clustered in time and were usually detected on 1–3 consecutive days with a median interval of 6.5 d for calls >1 d apart. Hourly calling rates were significantly higher at night than during the day. These data indicate that right whales occur in the SEBS later in the year than previously known, intermittently pass through the middle‐shelf study region, and usually remain there no longer than a few days. Right whale habitat use in the SEBS may intensify in mid‐summer through early fall based on higher monthly and daily call detection rates.     

Characterization of 14 polymorphic microsatellite loci for the lesser horseshoe bat,Rhinolophus hipposideros (Rhinolophidae,Chiroptera)

Fourteen polymorphic microsatellites isolated in the lesser horseshoe bat, Rhinolophus hipposideros, are described. A subset of eight loci successfully coamplified in a multiplex polymerase chain reaction (PCR), and allowed to confirm that expected heterozygosity was relatively high for the eight loci (0.56–0.83). This set of eight microsatellites indeed permits to build genetic tags that distinguish all individuals in colonies consisting of up to more than 1000 individuals.     

Development and multiplex PCR amplification of new microsatellite markers for the freshwater fish,ayu (Plecoglossus altivelis)

Multiplex polymerase chain reaction (PCR) technique has recently been applied to gain many advantages in molecular genetics. The present study focused on the development of 15 new microsatellite markers with multiplex PCR systems in ayu, Plecoglossus altivelis, an important freshwater fish in Japan. All loci were followed Mendelian inheritance in 27 F1 progeny except for the one locus. The number of alleles per locus ranged from nine to 44 and the observed heterozygosities ranged from 0.680 to 0.980 in 50 unrelated individuals. The results indicate that these new microsatellite markers are useful for studies of linkage mapping and population genetics for the species.     

Molecular identification of individual North Atlantic right whales (Eubalaena glacialis) using free-floating feces

During the 1990s, North Atlantic right whales had significantly decreased reproduction and showed signs of compromised health, prompting the initiation of noninvasive fecal-based studies to investigate potential causal factors. The interpretation of these studies is enhanced when the defecator is identified, as data can then be linked to individual life history information. Fecal samples (n= 118) were either collected from single photoidentified whales, associated with several individuals by photoidentification of whales in the vicinity upon sample collection, or were collected when no whales were in the vicinity. Genetic profiles from fecal DNA comprising sex, mitochondrial haplotype, and five microsatellite loci helped assign specific samples to individual right whales based on existing genetic profiles. Profiles were informative in assigning 61 fecal samples to known individuals, 24 of which were collected when no whales were in the vicinity. Whales identified genetically were typically photographed in the same habitat area and on the same day of sample collection (n= 35/48). Twelve profiles new to the genetic database were identified, suggesting fecal sampling provides a means to obtain genetic profiles from previously unsampled individuals, which may help refine estimates of population size and habitat use patterns if annual fecal sampling continues.     

Rope trauma,sedation, disentanglement,and monitoring‐tag associated lesions in a terminally entangled North Atlantic right whale (Eubalaena glacialis)

A chronically entangled North Atlantic right whale, with consequent emaciation was sedated, disentangled to the extent possible, administered antibiotics, and satellite tag tracked for six subsequent days. It was found dead 11 d after the tag ceased transmission. Chronic constrictive deep rope lacerations and emaciation were found to be the proximate cause of death, which may have ultimately involved shark predation. A broadhead cutter and a spring‐loaded knife used for disentanglement were found to induce moderate wounds to the skin and blubber. The telemetry tag, with two barbed shafts partially penetrating the blubber was shed, leaving barbs embedded with localized histological reaction. One of four darts administered shed the barrel, but the needle was found postmortem in the whale with an 80º bend at the blubber‐muscle interface. This bend occurred due to epaxial muscle movement relative to the overlying blubber, with resultant necrosis and cavitation of underlying muscle. This suggests that rigid, implanted devices that span the cetacean blubber muscle interface, where the muscle moves relative to the blubber, could have secondary health impacts. Thus we encourage efforts to develop new tag telemetry systems that do not penetrate the subdermal sheath, but still remain attached for many months.     

Estimated calf and perinatal mortality in western North Atlantic right whales (Eubalaena glacialis)

Reduced reproductive success has contributed to lack of recovery of the endangered western North Atlantic right whale (Eubalaena glacialis). Here we examined the specific life history period from just before birth through the first year to estimate calf and perinatal losses between 1989 and 2003. The lower bound estimate (17 mortalities from 208 calving events) included documented calf mortalities and presumed deaths from serious injury or disappearance from the sighting record. The upper bound estimated potential calf losses from females with delayed first parturition (>10 yr) and shortened (2 yr) or lengthened (≥4 yr) calving intervals, if the female migrated to the calving ground during these intervals. Because cows were sighted in the calving ground predominantly in years when they were available to calve, adult females sighted there in a possible calving year without a calf were assumed to have experienced a perinatal loss. Twenty-eight potential perinatal losses were detected, bringing the upper bound of calf and perinatal mortality to 45 (3.0 calves/yr). The high frequency of lengthened calving intervals in E. glacialis suggests that abortion and neonatal losses are contributing to lower reproductive success compared to Southern Hemisphere right whales (Eubalaena australis).     

Isolation and characterization of swift fox tetranucleotide microsatellite loci

Primers have been developed for nine polymorphic tetranucleotide microsatellite loci isolated from the swift fox (Vulpes velox). These can be amplified in three multiplex reactions and all loci can be scored easily and without ambiguity. The number of alleles per locus ranged from three to 14 and the overall heterozygosities ranged from 0.54 to 1.00. All loci were in Hardy‐Weinberg equilibrium, but two loci were in linkage disequilibrium. These loci will be used to monitor endangered swift fox populations on the Canadian prairies.     

Isolation of microsatellite loci in the freshwater goby,Rhinogobius sp. (Gobiidae)

The Rhinogobius species complex (Pisces: Gobiidae) includes great variation in colour, morphology and ecological form. In particular, R. sp. OR (Orange type) exhibits some of the greatest colour variation among the species complex, although the genetic relationships among the variations in this fish remain unclear. Thirteen microsatellite loci were identified from R. sp. OR, and all loci were polymorphic with two to 17 alleles per locus. Observed heterozygosity ranged from 0.1 to 0.9, while the expected heterozygosity ranged from 0.19 to 0.96. Multiplex polymerase chain reaction reaction (PCR) were optimized. All loci except Rhi‐5 conform to Hardy–Weinberg equilibrium (P > 0.05).     

Characterization of polymorphic tetranucleotide microsatellite loci from the Siberian weasel (Mustela sibirica)

Nine tetrarepeat microsatellite loci for Siberian weasel (Mustela sibirica) were characterized. By using 35 individuals of unknown relationship collected from Taiwan, we evaluated the polymorphism of these loci. The number of alleles for each locus ranged from five to 12 (mean = 8.78 alleles) and the observed heterozygosity from 0.429 to 0.962 (mean = 0.688) and all loci conformed to Hardy–Weinberg expectation.     

Characterization of 12 polymorphic microsatellite loci for eastern chipmunks (Tamias striatus)

Eastern chipmunks are small forest rodents for which ecological data at multiple spatial scales exist, but no molecular markers have been developed that provide adequate resolution to examine genetic structure at fine scales in this species. Thus, we developed 12 variable microsatellite loci, which amplified in three multiplexed reactions, and screened them in 27 individuals from northern Indiana, USA. Observed heterozygosities across loci ranged from 0.26 to 0.91 and number of alleles per locus ranged from three to 10. We will use these markers along with ecological data to understand population dynamics of eastern chipmunks at small spatial scales.     

Development of 14 multiplexed microsatellite loci for raccoons Procyon lotor

In order to complement ecological information with genetic data we isolated and characterized 14 polymorphic microsatellite markers from raccoons (Procyon lotor). Three multiplexed panels comprising the loci were developed and 29 individuals from a contiguous habitat patch in northern Indiana, USA were genotyped. The number of alleles per locus ranged from four to 18, and overall heterozygosities ranged from 0.31 to 1.00. One locus was identified as possibly being X‐linked, since males appeared to be hemizygous. Data generated using these markers will be used to further our understanding of small‐scale raccoon population dynamics in a highly fragmented landscape.