How Big Is It Really? Assessing the Efficacy of Indirect Estimates of Body Size in Asian Elephants

Information on an organism’s body size is pivotal in understanding its life history and fitness, as well as helping inform conservation measures. However, for many species, particularly large-bodied wild animals, taking accurate body size measurements can be a challenge. Various means to estimate body size have been employed, from more direct methods such as using photogrammetry to obtain height or length measurements, to indirect prediction of weight using other body morphometrics or even the size of dung boli. It is often unclear how accurate these measures are because they cannot be compared to objective measures. Here, we investigate how well existing estimation equations predict the actual body weight of Asian elephants Elephas maximus, using body measurements (height, chest girth, length, foot circumference and neck circumference) taken directly from a large population of semi-captive animals in Myanmar (n = 404). We then define new and better fitting formulas to predict body weight in Myanmar elephants from these readily available measures. We also investigate whether the important parameters height and chest girth can be estimated from photographs (n = 151). Our results show considerable variation in the ability of existing estimation equations to predict weight, and that the equations proposed in this paper predict weight better in almost all circumstances. We also find that measurements from standardised photographs reflect body height and chest girth after applying minor adjustments. Our results have implications for size estimation of large wild animals in the field, as well as for management in captive settings.     

Selective cross‐linking of coinciding protein assemblies by in‐gel cross‐linking mass spectrometry

Cross‐linking mass spectrometry has developed into an important method to study protein structures and interactions. The in‐solution cross‐linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross‐links obtained from co‐occurring protein oligomers, complexes, or conformers. Here we developed a cross‐linking workflow combining blue native PAGE with in‐gel cross‐linking mass spectrometry (IGX‐MS). This workflow circumvents steps, such as buffer exchange and cross‐linker concentration optimization. Additionally, IGX‐MS enables the parallel analysis of co‐occurring protein complexes using only small amounts of sample. Another benefit of IGX‐MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over‐length cross‐links compared to in‐solution cross‐linking. We next used IGX‐MS to investigate the complement components C5, C6, and their hetero‐dimeric C5b6 complex. The obtained cross‐links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX‐MS can provide new insights into the initial stages of the terminal complement pathway.     

Seychelles warblers with silver spoons: Juvenile body mass is a lifelong predictor of annual survival,but not annual reproduction or senescence

The environment experienced during development, and its impact on intrinsic condition, can have lasting outcomes for individual phenotypes and could contribute to variation in adult senescence trajectories. However, the nature of this relationship in wild populations remains uncertain, owing to the difficulties in summarizing natal conditions and in long‐term monitoring of individuals from free‐roaming long‐lived species. Utilizing a closely monitored, closed population of Seychelles warblers (Acrocephalus sechellensis), we determine whether juvenile body mass is associated with natal socioenvironmental factors, specific genetic traits linked to fitness in this system, survival to adulthood, and senescence‐related traits. Juveniles born in seasons with higher food availability and into smaller natal groups (i.e., fewer competitors) were heavier. In contrast, there were no associations between juvenile body mass and genetic traits. Furthermore, size‐corrected mass—but not separate measures of natal food availability, group size, or genetic traits—was positively associated with survival to adulthood, suggesting juvenile body mass is indicative of natal condition. Heavier juveniles had greater body mass and had higher rates of annual survival as adults, independent of age. In contrast, there was no association between juvenile mass and adult telomere length attrition (a measure of somatic stress) nor annual reproduction. These results indicate that juvenile body mass, while not associated with senescence trajectories, can influence the likelihood of surviving to old age, potentially due to silver‐spoon effects. This study shows that measures of intrinsic condition in juveniles can provide important insights into the long‐term fitness of individuals in wild populations.     

Temporal change in the morphometry–body mass relationship of polar bears

Accurate information on animal body mass is often an essential component of wildlife research and management. However, for many large-bodied species, obtaining direct scale weights from individuals may be difficult. In these cases, morphometric equations (e.g., based on girth or length) may provide accurate and precise estimates of body mass. We developed predictive equations to estimate the body mass of free-ranging polar bears (Ursus maritimus) in western Hudson Bay, Canada. Using multiple linear and non-linear regression, we identified a strong relationship between polar bear body weight and linear measures of straight line length and axillary girth. The mass–morphometry relationship appeared to change over time and we developed separate equations for polar bears measured during 2 time periods, 1980–1996 and 2007–2009. Non-linear models were more accurate and provided body mass estimates within 5.8% (R² = 0.98) and 6.1% (R² = 0.98) of scale weight in the earlier and later time periods, respectively. Earlier equations developed for polar bears in this subpopulation performed poorly when applied to recently sampled individuals. In contrast, some contemporary equations from other regions performed reasonably well, suggesting that temporal changes within a subpopulation may be more pronounced than regional differences and can render earlier predictive equations obsolete. Our results have important implications for current and future studies of polar bear body condition and the effects of ongoing climate warming. © 2011 The Wildlife Society.     

Methodological confounds of measuring urinary oxidative stress in wild animals

Biomarkers of oxidative stress (OS) are useful in addressing a wide range of research questions, but thus far, they have had limited application to wild mammal populations due to a reliance on blood or tissue sampling. A shift toward non‐invasive measurement of OS would allow field ecologists and conservationists to apply this method more readily. However, the impact of methodological confounds on urinary OS measurement under field conditions has never been explicitly investigated. We combined a cross‐sectional analysis with a field experiment to assess the impact of four potential methodological confounds on OS measurements: (1) time of sampling, (2) environmental contamination from foliage; (3) delay between sample collection and flash‐freezing in liquid nitrogen; and (4) sample storage of up to 15 months below −80°C. We measured DNA oxidative damage (8‐hydroxy‐2′‐deoxyguanosine, 8‐OHdG), lipid peroxidation (malondialdehyde, MDA), total antioxidant capacity (TAC), and uric acid (UA) in 167 urine samples collected from wild Zanzibar red colobus (Piliocolobus kirkii). We found that MDA was higher in samples collected in the morning than in the afternoon but there were no diurnal patterns in any of the other markers. Contamination of samples from foliage and length of time frozen at −80°C for up to 15 months did not affect OS marker concentrations. Freezing delay did not affect OS levels cross‐sectionally, but OS values from individual samples showed only moderate‐to‐good consistency and substantial rank‐order reversals when exposed to different freezing delays. We recommend that diurnal patterns of OS markers and the impact of storage time before and after freezing on OS marker concentrations be considered when designing sampling protocols. However, given the high stability we observed for four OS markers subject to a variety of putative methodological confounds, we suggest that urinary OS markers provide a valuable addition to the toolkit of field ecologists and conservationists within reasonable methodological constraints.     

A method to evaluate body length of live aquatic vertebrates using digital images

Traditional methods to measure body lengths of aquatic vertebrates rely on anesthetics, and extended handling times. These procedures can increase stress, potentially affecting the animal''s welfare after its release. We developed a simple procedure using digital images to estimate body lengths of coastal cutthroat trout (Oncorhynchus clarkii clarkii) and larval coastal giant salamander (Dicamptodon tenebrosus). Images were postprocessed using ImageJ2. We measured more than 900 individuals of these two species from 200 pool habitats along 9.6 river kilometers. The percent error (mean ± SE) of our approach compared to the use of a traditional graded measuring board was relatively small for all length metrics of the two species. Total length of trout was −2.2% ± 1.0. Snout–vent length and total length of larval salamanders was 3.5% ± 3.3 and −0.6% ± 1.7, respectively. We cross‐validated our results by two independent observers that followed our protocol to measure the same animals and found no significant differences (p > .7) in body size distributions for all length metrics of the two species. Our procedure provides reliable information of body size reducing stress and handling time in the field. The method is transferable across taxa and the inclusion of multiple animals per image increases sampling efficiency with stored images that can be reviewed multiple times. This practical tool can improve data collection of animal size over large sampling efforts and broad spatiotemporal contexts.     

Validating the use of stereo‐video cameras to conduct remote measurements of sea turtles

Point 1: Stereo‐video camera systems (SVCSs) are a promising tool to remotely measure body size of wild animals without the need for animal handling. Here, we assessed the accuracy of SVCSs for measuring straight carapace length (SCL) of sea turtles.Point 2: To achieve this, we hand captured and measured 63 juvenile, subadult, and adult sea turtles across three species: greens, Chelonia mydas (n = 52); loggerheads, Caretta caretta (n = 8); and Kemp''s ridley, Lepidochelys kempii (n = 3) in the waters off Eleuthera, The Bahamas and Crystal River, Florida, USA, between May and November 2019. Upon release, we filmed these individuals with the SVCS. We performed photogrammetric analysis to extract stereo SCL measurements (eSCL), which were then compared to the (manual) capture measurements (mSCL).Point 3: mSCL ranged from 25.9 to 89.2 cm, while eSCL ranged from 24.7 to 91.4 cm. Mean percent bias of eSCL ranged from −0.61% (±0.11 SE) to −4.46% (±0.31 SE) across all species and locations. We statistically analyzed potential drivers of measurement error, including distance of the turtle to the SVCS, turtle angle, image quality, turtle size, capture location, and species.Point 4: Using a linear mixed effects model, we found that the distance between the turtle and the SVCS was the primary factor influencing measurement error. Our research suggests that stereo‐video technology enables high‐quality measurements of sea turtle body size collected in situ without the need for hand‐capturing individuals. This study contributes to the growing knowledge base that SVCS are accurate for body size measurements independent of taxonomic clade.     

Exploring the Genetic Signature of Body Size in Yucatan Miniature Pig

Since being domesticated about 10,000–12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig.     

Morphometric variation among river sturgeons (Scaphirhynchus spp.) of the Middle and Lower Mississippi River

Pallid sturgeon (Scaphirhynchus albus) captured in the Middle and Lower Mississippi River (i.e. below St. Louis, MO, USA) are morphologically very similar to shovelnose sturgeon (Scaphirhynchus platorynchus). Available empirical data are limited to a few studies based on low sample sizes from disjointed populations. Geneticists are currently searching for markers that will differentiate the two species, but the need for unequivocal species‐specific field characters remains. Continuation of commercial fishing for shovelnose sturgeon in some states necessitates an immediate means for accurate field identifications. Previous studies of lower basin river sturgeon classified individuals with simple morphometric character indices and interpreted intermediacy as interspecific hybridization. In this study, morphometric variation among Scaphirhynchus specimens from the Middle and Lower Mississippi River is examined for evidence of hybridization. Data are compared for large (>250‐mm standard length) hatchery‐reared and wild pallid specimens and wild shovelnose specimens. Specimens are compared using two morphometric character indices, two morphometric/meristic character indices and principal components analysis. Results indicate substantial morphological variation among pallid sturgeon below the mouth of the Missouri River. The amount of variation appears to decrease downstream in the Mississippi River. Sheared principal components analysis of morphometric data shows complete separation of shovelnose and pallid sturgeon specimens, whereas character indices indicate overlap. Both character indices and sheared principal components analysis demonstrate that pallid sturgeon in the Lower Mississippi River are morphologically more similar to shovelnose sturgeon than are pallids from the Upper Missouri River. This similarity, explained in previous studies as hybridization, may be the result of latitudinal morphometric variation and length‐at‐age differences between populations of the upper and lower extremes of the range.     

Measuring changes in physical size and predicting weight of sows during gestation

Changes in physical body size during gestation were monitored using 529 sets of sow measurements. All sows were from the same herd and production system with a range in parity from 1 to 8. Sows were individually weighed, P2 backfat thickness was determined by ultrasound and morphometric measurements of body size were taken five times during gestation: day 0 (at service), day 25, day 50, day 80 and day 110. The morphometric measurements included sow height (from floor to last rib at the midline, from floor to ventral surface and from floor to hip), heart girth, depth of last rib, length (from snout to tail and from anterior scapula to tail) and width (at ham, at last rib and at shoulder). Regression analyses were used to model the relationship between day of gestation or parity number and morphometric measurements of body size. Regression equations were also developed to estimate sow weight from physical measurements, day of gestation and parity. As expected, sow dimensions, in general, increased as pregnancy progressed and also with increasing parity number. The relationships between day of gestation and body dimensions were described by linear and quadratic regression models, which had a range of adjusted R2 values up to 0.99. Similar relationships to parity number had a range of R2 values between 0.51 and 0.96. Sow depth, which can be used as an estimate of the width of the sow when lying, equalled the maximum width of the gestation stall (650 mm) at day 103 of gestation. However, by day 40 of gestation, predicted mean sow depth (570 mm) equalled the width at the rear of the crate. The implication of this is that after day 40 of gestation, the average sow was too wide for the rear of the crate when lying in a recumbent position. On day 110 of gestation, 95% of the mean sow body depths would be accommodated in stalls that were 674 mm wide; however, the range in body sizes with increasing parity number suggests the use of more than one stall width would be appropriate. Sow weight could be estimated with an adjusted R2 value of 0.81 and with a residual standard deviation (r.s.d.) of 16.5 kg using heart girth alone, or more accurately using a model with parity, day of gestation, P2 backfat depth and heart girth as the parameters (R2 = 0.89, r.s.d. 12.4 kg).     

Advancing the use of morphometric data for estimating and managing common bottlenose dolphin (Tursiops truncatus) mass

Knowledge of a dolphin's body mass is central to establishing body condition, comparing across individuals, and designing successful management programs. In the present study, sex‐specific prediction equations for estimating body mass were generated from morphometrics (i.e., length and girth) and ages of bottlenose dolphins residing under professionally managed care. Measurements of wild dolphins in Sarasota Bay, Florida, were used to generate sex‐specific body mass reference ranges. Gompertz growth models were fitted to length measurements and age to compare growth across populations. From the regression analyses, the body mass of managed females (R² = 0.937), managed males (R² = 0.953), wild females (R² = 0.979), and wild males (R² = 0.972) were predicted with high levels of accuracy. Managed adults had similar or longer asymptotic lengths compared to their wild conspecifics. To apply this information, ZooMorphTrak, a mobile software application, was developed to provide a new resource for management. The “Approximate” feature was designed to approximate body mass based on user inputs of individual morphometrics. The “Management” feature compared a managed dolphin's known body mass with respect to body mass reference ranges generated from wild dolphins. ZooMorphTrak, developed by the Chicago Zoological Society, is available for download at http://itunes.apple.com .     

Applicability of Single-Camera Photogrammetry to Determine Body Dimensions of Pinnipeds: Galapagos Sea Lions as an Example

Morphological features correlate with many life history traits and are therefore of high interest to behavioral and evolutionary biologists. Photogrammetry provides a useful tool to collect morphological data from species for which measurements are otherwise difficult to obtain. This method reduces disturbance and avoids capture stress. Using the Galapagos sea lion (Zalophus wollebaeki) as a model system, we tested the applicability of single-camera photogrammetry in combination with laser distance measurement to estimate morphological traits which may vary with an animal’s body position. We assessed whether linear morphological traits estimated by photogrammetry can be used to estimate body length and mass. We show that accurate estimates of body length (males: ±2.0%, females: ±2.6%) and reliable estimates of body mass are possible (males: ±6.8%, females: 14.5%). Furthermore, we developed correction factors that allow the use of animal photos that diverge somewhat from a flat-out position. The product of estimated body length and girth produced sufficiently reliable estimates of mass to categorize individuals into 10 kg-classes of body mass. Data of individuals repeatedly photographed within one season suggested relatively low measurement errors (body length: 2.9%, body mass: 8.1%). In order to develop accurate sex- and age-specific correction factors, a sufficient number of individuals from both sexes and from all desired age classes have to be captured for baseline measurements. Given proper validation, this method provides an excellent opportunity to collect morphological data for large numbers of individuals with minimal disturbance.     

Assessment of body condition of harp seals

We investigated the relationships between blubber content and a suite of condition indices in harp seals (Phoca groenlandica). Although blubber content was significantly correlated with xiphosternal blubber depth and Condition index they were insensitive indicators of condition. Blubber depth was most variable at a site ca. 60–70% down the length of the body and a more recent index incorporating length, mass and blubber depth devised for phocids was an improved indicator of blubbler mass. Blubber volume was calculated in three ways based on a geometric approximation of a cylinder and also via truncated cones. The cone method accounted for more variance than the other methods although it overestimated blubber mass. Surface area was ascertained using the girth measurements of serial cones and was significantly correlated with total body mass and total body water. We suggest that estimates of total body water generated from surface area could be used to calculate total fat mass and lean body mass     

Modeling and Mapping the Probability of Occurrence of Invasive Wild Pigs across the Contiguous United States

Wild pigs (Sus scrofa), also known as wild swine, feral pigs, or feral hogs, are one of the most widespread and successful invasive species around the world. Wild pigs have been linked to extensive and costly agricultural damage and present a serious threat to plant and animal communities due to their rooting behavior and omnivorous diet. We modeled the current distribution of wild pigs in the United States to better understand the physiological and ecological factors that may determine their invasive potential and to guide future study and eradication efforts. Using national-scale wild pig occurrence data reported between 1982 and 2012 by wildlife management professionals, we estimated the probability of wild pig occurrence across the United States using a logistic discrimination function and environmental covariates hypothesized to influence the distribution of the species. Our results suggest the distribution of wild pigs in the U.S. was most strongly limited by cold temperatures and availability of water, and that they were most likely to occur where potential home ranges had higher habitat heterogeneity, providing access to multiple key resources including water, forage, and cover. High probability of occurrence was also associated with frequent high temperatures, up to a high threshold. However, this pattern is driven by pigs’ historic distribution in warm climates of the southern U.S. Further study of pigs’ ability to persist in cold northern climates is needed to better understand whether low temperatures actually limit their distribution. Our model highlights areas at risk of invasion as those with habitat conditions similar to those found in pigs’ current range that are also near current populations. This study provides a macro-scale approach to generalist species distribution modeling that is applicable to other generalist and invasive species.     

Estimation of body fat in female rhesus monkeys

Measurement of height (crown–rump length), body weight, and abdominal subcutaneous fat depth, based on skinfold thickness taken from 13 female rhesus monkeys comprising two age groups were correlated with body fat values derived from tritiated water determinations of total body water. The manner with which each measure was related to percent body fat differed as a function of age of the animal. In the young, nulliparous females, crown–rump length was the single best predictor of body fat, whereas in the older, multiparous females, skinfold thickness correlated most highly with body fat. When all measurements, including the Quetelet index [(wt/ht)² × 1,000], were combined statistically and regressed against percent body fat, a significant increase in predictive ability was obtained. When each age group was considered separately, the resulting equations again reflected the agegroup biases. In addition, as an internal check on the validity of the regression equations, an additional regression analysis was performed using morphometric data from selected animals in each age group. These equations yielded accurate estimates of body fat when compared to determinations made from total body water. These analyses indicate that the predictive accuracy of morphometric data is greatly enhanced by using these measurements in concert. Furthermore, the utility of such predictions is influenced by the specific physical characteristics of the subject population.     

Great tits do not compensate over time for a radio‐tag‐induced reduction in escape‐flight performance

The use of biologging and tracking devices is widespread in avian behavioral and ecological studies. Carrying these devices rarely has major behavioral or fitness effects in the wild, yet it may still impact animals in more subtle ways, such as during high power demanding escape maneuvers. Here, we tested whether or not great tits (Parus major) carrying a backpack radio‐tag changed their body mass or flight behavior over time to compensate for the detrimental effect of carrying a tag. We tested 18 great tits, randomly assigned to a control (untagged) or one of two different types of a radio‐tag as used in previous studies in the wild (0.9 g or 1.2 g; ~5% or ~6–7% of body mass, respectively), and determined their upward escape‐flight performance 1, 7, 14, and 28 days after tagging. In between experiments, birds were housed in large free‐flight aviaries. For each escape‐flight, we used high‐speed 3D videography to determine flight paths, escape‐flight speed, wingbeat frequency, and actuator disk loading (ratio between the bird weight and aerodynamic thrust production capacity). Tagged birds flew upward with lower escape‐flight speeds, caused by an increased actuator disk loading. During the 28‐day period, all groups slightly increased their body mass and their in‐flight wingbeat frequency. In addition, during this period, all groups of birds increased their escape‐flight speed, but tagged birds did so at a lower rate than untagged birds. This suggests that birds may increase their escape‐flight performance through skill learning; however, tagged birds still remained slower than controls. Our findings suggest that tagging a songbird can have a prolonged effect on the performance of rapid flight maneuvers. Given the absence of tag effects on reproduction and survival in most songbird radio‐tagging studies, tagged birds in the wild might adjust their risk‐taking behavior to avoid performing rapid flight maneuvers.     

ESTIMATION OF BODY MASS IN THE SOUTHERN ELEPHANT SEAL, MIROUNGA LEONINA, BY PHOTOGRAMMETRY AND MORPHOMETRICS

A simple photographic technique was developed to indirectly estimate body mass data for southern elephant seal ( Mirounga leonina ) cows (postlactation), yearlings, and immature males and females. Regressions of mass on both photographic and morphometric variables (together and separately) yielded useful, predictable models. Using such variables, the best estimation of the actual mass was for postlactation cows, with a 95% confidence interval of ± 2.66% of the predicted body mass. Although combining photographic and morphometric variables produced the most reliable models specifically for cows and yearlings, the most practical model contained only the morphometric variables length and girth squared. Side area was the best correlated single photographic variable and this corresponded with other studies. Photogrammetry could be useful when animals cannot be sedated and are located on a flat surface, but it does require animals to be motionless when approached. Thus, the procedure may be more suited to bulls rather than other age classes and could have a role in studies where large numbers of mass estimations are rapidly required. If sedation is utilized in smaller animals, then the use of body length and girth is the most suitable indirect mass estimation technique to avoid the use of heavy weighing equipment.     

Influence of plant reproductive systems on the evolution of hummingbird pollination

Many hummingbird‐pollinated plant species evolved from bee‐pollinated ancestors independently in many different habitats in North and South America. The mechanisms leading to these transitions are not completely understood. We conducted pollination and germination experiments and analyzed additional reproductive traits in three sister species pairs of which one species is bee‐ and the other hummingbird‐pollinated. All hummingbird‐pollinated species showed higher seed set and germination rates in cross‐pollinated than in self‐pollinated flowers. In the self‐compatible, bee‐pollinated sister species this difference did not exist. As expected, seed set and germination rate were higher after cross‐pollination in the largely self‐incompatible genus Penstemon independently of the pollination syndrome. However, the bird‐pollinated species produce only half of the amount of ovules and pollen grains per flower compared to the bee‐pollinated sister species. This indicates that hummingbird pollination is much more efficient in self‐incompatible populations because hummingbirds waste less pollen and provide higher outcrossing rates. Therefore, hummingbird pollination is less resource costly. Overall, we suggest that hummingbirds may increase the reproductive success compared to bees, influencing the evolution of hummingbird pollination in ecosystems with diverse bee assemblages.     

Morphometric study of Kalophrynus palmatissimus at two forest reserves in Malaysia

A research study on morphometrics of Kalophrynus palmatissimus (commonly known as Lowland Grainy Frog) at Ayer Hitam Forest Reserve (AHFR), Selangor and Pasoh Forest Reserve (PFR), Negeri Sembilan was carried out from 12 November 2016 to 13 September 2017. The study was to examine data on the morphometric traits of K. palmatissimus at the two forest reserves. 15 morphometric traits of K. palmatissimus that were taken by using vernier calipers. Frog surveys were done by using 15 and 18 nocturnal 400 m transect lines with an interval distance of 20 m at AHFR and PFR, respectively. The GPS coordinates for all frog samples were recorded to ensure the precise geographic location. In addition, five climatic data were recorded. The results showed that most morphometric traits in AHFR (n = 34) and PFR (n = 31) were positively correlated with each other. On the other hand, climatic factor, which was soil pH, had a significant positive influence on most of the morphometric traits (p < .01), except for tympanum diameter and upper eyelid width (p ≥ .05). Meanwhile, the temperature had a significantly negative influence on all morphometric traits (p < .01). General linear model (GLM) analysis showed that snout‐vent length (SVL) influenced most morphometric traits (F ≤ 80.86, p < .01), except for hand length (HAL: F = 0.299, p > .05). Later, it was found that the snout‐vent length of K. palmatissimus at AHFR was slightly larger than at PFR (AHFR: μ = 37.00 mm, SE = 1.16 c.f. PFR: μ = 30.29 mm, SE = 1.07). It showed that there were variations in morphometric traits of K. palmatissimus at AHFR and PFR. From PCA analysis, morphometric traits are grouped into two components for AHFR and PFR, respectively. In AHFR, head length, eye diameter, head width, internarial distance, interorbital distance, forearm length, tibia length, foot length, and thigh length were strongly correlated, while snout length and eye‐nostril distance were strongly correlated. In PFR, eye diameter, head width, internarial distance, interorbital distance, foot length, and thigh length were strongly correlated, though snout length and eye‐nostril distance were strongly correlated, hence, suggested that all morphometric traits grow simultaneously in K. palmatissimus with eye‐nostril distance (EN), and snout length (SL) growing almost simultaneously at AHFR (r = .91) and PFR (r = .97). There is still a lack of available information regarding the distribution and morphometric studies of K. palmatissimus in Malaysia, especially at AHFR and PFR. This study showed 15 different morphometric traits of K. palmatisssimus between AHFR and PFR, with K. palmatissimus at AHFR were found to be slightly larger than at PFR.     

Modification of the N‐terminal FWKG−αH1 element of potyviral HC‐Pro affects its multiple functions and generates effective attenuated mutants for cross‐protection

Control of plant viruses by cross‐protection is limited by the availability of effective protective strains. Incorporation of an NIa‐protease processing site in the extreme N‐terminal region of the helper component protease (HC‐Pro) of turnip mosaic virus (TuMV) resulted in a mutant virus TuHN^DI that induced highly attenuated symptoms. Recombination analysis verified that two variations, F7I mutation and amino acid 7‐upstream‐deletion, in HC‐Pro co‐determined TuHN^DI attenuation. TuHN^DI provided complete protection to Nicotiana benthamiana and Brassica campestris subsp. chinensis plants against infection by the severe parental strain. Aphid transmission tests revealed that TuHN^DI was not aphid‐transmissible. An RNA silencing suppression (RSS) assay by agroinfiltration suggested the RSS‐defective nature of the mutant HC‐Pro. In the context (amino acids 3–17) encompassing the two variations of HC‐Pro, we uncovered an FWKG−α‐helix 1 (αH1) element that influenced the functions of aphid transmission and RSS, whose motifs were located far downstream. We further demonstrated that HC‐Pro F7 was a critical residue on αH1 for HC‐Pro functions and that reinstating αH1 in the RSS‐defective HC‐Pro of TuHN^DI restored the protein''s RSS function. Yeast two‐hybrid and bimolecular fluorescence complementation assays indicated the FWKG−αH1 element as an integral part of the HC‐Pro self‐interaction domain. The possibility of regulation of the mechanistically independent functions of RSS and aphid transmission by the FWKG−αH1 element is discussed. Extension of TuMV HC‐Pro FWKG−αH1 variations to another potyvirus, zucchini yellow mosaic virus, also generated nonaphid‐transmissible cross‐protective mutant viruses. Hence, the modification of the FWKG−αH1 element can generate effective attenuated viruses for the control of potyviruses by cross‐protection.