Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae)

The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, SspI (M. gouazoubira) and AflIII (M. americana, and M. nana).     

The red brocket deer (Mazama americana) as a new intermediate host of Taenia omissa (Taeniidae)

A total of 13 metacestodes were collected from the lung and parietal pleura from a red brocket deer (Mazama americana) from the Peruvian Amazon. All metacestodes were identified as cysticerci of Taenia omissa by morphological and molecular analyzes. Cytochrome c oxidase subunit 1 sequences from the new isolate T. omissa had more than 96.8% identity with other Peruvian isolates of the species previously sequenced. Lower similarities (93.8–95.8%) were verified between Peruvian and Canadian isolates. This finding adds a new intermediate host for T. omissa and also expands its geographical distribution.     

Differential Responses to Hunting in Two Sympatric Species of Brocket Deer (Mazama americana and M. nana)

Hunting by humans may affect the abundance and activity patterns of game species. We examined the effect of hunting on the abundance and activity patterns of sympatric red brocket deer Mazama americana and dwarf brocket deer M. nana . We conducted four camera-trap surveys (158 sampling stations, 10,244 trap-days, total area sampled 1200 km²) in three areas within the Atlantic Forest of Misiones, Argentina, that differ in protection and hunting pressure. We used logistic regression and tests of independence to evaluate if protection, hunting pressure, and other independent variables affect the probability of recording each species and their recording rate. We used the Mardia–Watson–Wheeler test to examine if the daily activity pattern differs between species and changes with hunting pressure. Red brocket deer were more frequently recorded (397 records, 58% of stations) than dwarf brocket deer (100 records, 37% of stations). The probability of recording red brockets was higher in areas with better protection and increased with the distance to the main accesses used by poachers. The probability of recording dwarf brockets was higher in areas with low protection. Red brockets were more nocturnal than dwarf brockets, a difference that may reduce interspecific competition. However, red brockets were more diurnal in the best-protected areas, suggesting that they can adjust their activity to local hunting pressure. Hunting has opposite effects on the abundance of these deer and may facilitate their coexistence. Hunting should be carefully controlled or managed to ensure the conservation of these little known species.     

Characterization of the complete mitochondrial genome and a set of polymorphic microsatellite markers through next-generation sequencing for the brown brocket deer Mazama gouazoubira

The complete mitochondrial genome of the brown brocket deer Mazama gouazoubira and a set of polymorphic microsatellite markers were identified by 454-pyrosequencing. De novo genome assembly recovered 98% of the mitochondrial genome with a mean coverage of 9-fold. The mitogenome consisted of 16,356 base pairs that included 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and the control region, as found in other deer. The genetic divergence between the mitogenome described here and a previously published report was ∼0.5%, with the control region and ND5 gene showing the highest intraspecific variation. Seven polymorphic loci were characterized using 15 unrelated individuals; there was moderate genetic variation across most loci (mean of 5.6 alleles/locus, mean expected heterozygosity = 0.70), with only one locus deviating significantly from Hardy-Weinberg equilibrium, probably because of null alleles. Marker independence was confirmed with tests for linkage disequilibrium. The genetic variation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the phylogeography and population genetic patterns in M. gouazoubira, particularly in the context of habitat fragmentation in South America.     

Olfactory communication and counter-marking in brown brocket deer <Emphasis Type="Italic">Mazama gouazoubira</Emphasis>

Scent marking by defecation and urination in numerous small latrines may be related to resource defence in brown brocket deer (Mazama gouazoubira). Both males and females seem to be territorial, and both contribute to latrines where their ranges overlap. Latrines could thus potentially function as centres of information exchange and intrasexual competition. Counter-marking occurs when animals respond to invaders' marks with a greater number of marks. The objectives of this experimental study were to determine whether brown brocket deer distinguish dung of presumed invaders from their own and whether they counter-mark such faecal deposits. Two samples of dung (from unknown males or females and the experimental animal) were introduced near the latrines of 21 captive deer (13 males and eight females), and we observed their responses, including investigative (sniffing) and marking (urination and defecation) behaviours. Males investigated the introduced dung and their own latrine significantly more when the dung was from an unknown male than when it was their own. Females investigated unknown female dung significantly more than their own. Males counter-marked the introduced dung and their latrine significantly more when the dung was from an unknown male than when it was their own. Males marked with a shorter latency and at a greater frequency than females. Our data indicated that males counter-mark most intensively dung from male ‘intruders’ which may be related to intrasexual competition and resource defence. Females showed a non-significant tendency to counter-mark same sex intruders.     

The Taxonomic Status of Mazama bricenii and the Significance of the Táchira Depression for Mammalian Endemism in the Cordillera de Mérida,Venezuela

We studied the taxonomy and biogeography of Mazama bricenii, a brocket deer classified as Vulnerable by the IUCN, drawing on qualitative and quantitative morphology and sequences of the mitochondrial cytochrome-b gene. We used Ecological Niche Modeling (ENM) to evaluate the hypothesis that M. bricenii of the Venezuelan Cordillera de Mérida (CM) might have become isolated from populations of its putative sister species, Mazama rufina, in the Colombian Cordillera Oriental (CO). This hypothesis assumes that warm, dry climatic conditions in the Táchira Depression were unsuitable for the species. Our analyses did not reveal morphological differences between specimens geographically attributable to M. bricenii and M. rufina, and phylogenetic analyses of molecular data recovered M. bricenii nested within the diversity of M. rufina. These results indicate that M. bricenii should be regarded as a junior synonym of M. rufina. ENM analyses revealed the existence of suitable climatic conditions for M. rufina in the Táchira Depression during the last glacial maximum and even at present, suggesting that gene flow between populations in the CO and CM may have occurred until at least the beginning of the current interglacial period and may continue today. Because this pattern might characterize other mammals currently considered endemic to the CM, we examined which of these species match two criteria that we propose herein to estimate if they can be regarded as endemic to the CM with confidence: (1) that morphological or molecular evidence exists indicating that the putative endemic taxon is distinctive from congeneric populations in the CO; and (2) that the putative endemic taxon is restricted to either cloud forest or páramo, or both. Only Aepeomys reigi, Cryptotis meridensis, and Nasuella meridensis matched both criteria; hence, additional research is necessary to assess the true taxonomic status and distribution of the remaining species thought to be CM endemics.     

Influence of digestive morphology on resource partitioning in Amazonian ungulates

Summary Resource partitioning of diet and habitat use was studied in the entire Amazonian ungulate community of Northeastern Peru, which comprises the red brocket deer (Mazama americana), grey brocket deer (M. gouazoubira), collared peccary (Tayassu tajacu), white-lipped peccary (T. pecari), and lowland tapir (Tapirus terrestris). Each ungulate species partitioned at least one type of resource from every other species. Digestive morphology had a greater influence on resource partitioning of diet than body size. Neither digestive morphology nor body size were related to segregation of habitats. However, species with similar diets partitioned habitats, whereas species with different diets often used the same type of forest. Increases in habitat breadth of ungulates were positively correlated with increases in dietary breadth.     

Relative DNA Content and Chromosomal Relationships of some Meloidogyne, Heterodera, and Meloidodera spp. (Nematoda: Heteroderidae)

The relative DNA content of hypodermal nuclei of preparasitic, 2nd-stage larvae was determined cytophotometrically in 19 populations belonging to 13 species of Meloidogyne, Heterodera and Meloidodera. In Meloidogyne hapla, M. arenaria, M. incognita and M. javanica, total DNA content per nucleus is proportional to their chromosome number, indicating that chromosomal forms with high chromosome numbers are truly polyploid. M. graminicola, M. grarninis and M. ottersoni have a DNA content per chromosome significantly lower than that of the other Meloidogyne species. Within Heterodera, species with high chromosome numbers have proportionally higher DNA content, indicating again polyploidy. DNA content per chromosome in Meloidogyne is one third that of Heterodera and one haft that of Meloidodera floridensis. The karyotypic relationships of the three genera are still not clearly understood.     

Estimation of Genetic Divergence in Meloidogyne Mitochondrial DNA

Restriction fragments from purified mitochondrial DNA can be readily detected following rapid end-labeling with [α-³²]nucleoside triphosphates and separation by gel electrophoresis. Mitochondrial DNA from 12 populations of Meloidogyne species was digested with 12 restriction enzymes producing more than 60 restriction fragments for each species. The mitochondrial genome of M. arenaria is the most genetically distinct of the four species compared. M. arenaria shows approximately 2.1-3.1% nucleotide sequence divergence from the mitochondrial genomes of M. javanica, M. incognita, and M. hapla. Among the latter three species, interspecific estimates of sequence divergence range from 0.7 to 2.3%. Relatively high intraspecific variation in mitochondrial restriction fragment patterns was observed in M. hapla. Intraspecific variation in M. incognita resulted in sequence divergence estimates of 0.5-1.0%. Such polymorphisms can serve as genetic markers for discerning mitochondrial DNA genotypes in nematode populations in the same way that allozymes have been used to discern nuclear DNA genotypes.     

The melectine bee genera Brachymelecta and Sinomelecta (Hymenoptera,Apidae)

The enigmatic, cleptoparasitic bee genera Brachymelecta Linsley and Sinomelecta Baker (Apinae: Melectini) are redescribed, each represented by a single species which has not been reencountered since capture of the type series ca. 1878 and 1900, respectively. Both genera are the only melectines to possess two submarginal cells in the forewing but are otherwise wholly dissimilar. Brachymelecta mucida (Cresson), a species known only from the male holotype collected in “Nevada”, is newly described and figured, including the first account of the hidden sterna and genitalia. Sinomelecta oreina Baker is similarly described and figured based on the holotype male and paratype female, apparently collected from the eastern Tibetan Plateau. Both genera are valid and from the available data do not appear to represent merely autapomorphic forms of Melecta Latreille. Indeed, the terminalia of Sinomelecta oreina are in some respects more similar to those of species of Thyreus Panzer.     

Insecticidal effectiveness ofMammea Americana (Guttiferae) extracts on larvae ofDlabrotica Virgifera Virgifera (Coleoptera:Chrysomelidae) andTrichoplusia Ni (Lepidoptera:Noctuidae)

Seed and leaf extracts ofMammea americana (mamey apple) have a historical use as a biopesticide with the active components previously characterized. We reexamined the utility of this natural bioinsecticide in light of existing sources of material as a by-product of the fruit processing industry. Our results addDiabrotica virgifera virgifera andTrichoplusia ni to the list of insects which are susceptible to the insecticidal ingredients ofM. americana and confirms earlier reports of activity againstBlatella germanica, Periplaneta americana, andPlutella xylostella. We report LD5Os for crude hexane extracts ofM. americana leaves and seeds againstT. ni. These materials represent renewable sources of bioinsecticides for agriculture, and should regenerate interest in coumarin-type compounds for novel pesticidal action.     

Morphology of the larvae,male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.     

Comparison between Colony Morphology and Molecular Phylogeny in the Caribbean Scleractinian Coral Genus Madracis

A major challenge in coral biology is to find the most adequate and phylogenetically informative characters that allow for distinction of closely related coral species. Therefore, data on corallite morphology and genetic data are often combined to increase phylogenetic resolution. In this study, we address the question to which degree genetic data and quantitative information on overall coral colony morphologies identify similar groupings within closely related morphospecies of the Caribbean coral genus Madracis. Such comparison of phylogenies based on colony morphology and genetic data will also provide insight into the degree to which genotype and phenotype overlap. We have measured morphological features of three closely related Caribbean coral species of the genus Madracis (M. formosa, M. decactis and M. carmabi). Morphological differences were then compared with phylogenies of the same species based on two nuclear DNA markers, i.e. ATPSα and SRP54. Our analysis showed that phylogenetic trees based on (macroscopical) morphological properties and phylogenetic trees based on DNA markers ATPSα and SRP54 are partially similar indicating that morphological characteristics at the colony level provide another axis, in addition to commonly used features such as corallite morphology and ecological information, to delineate genetically different coral species. We discuss this new method that allows systematic quantitative comparison between morphological characteristics of entire colonies and genetic data.     

Identification of Single Meloidogyne Juveniles by Polymerase Chain Reaction Amplification of Mitochondrial DNA

Polymerase chain reaction (PCR) was used to amplify a specific 1.8-kb sequence of mitochondrial DNA from single juveniles and eggs from 17 populations of Meloidogyne incognita, M. hapla, M. javanica, and M. arenaria. Approximately 2 μg amplified product were produced per reaction. Restriction digestion of the amplified product with HinfI permitted discrimination of clonal lineages of the four species. Meloidogyne javanica, however, could not be separated from M. hapla by the enzymes used in these experiments. Various amplification conditions and nematode lysis procedures were examined in order to optimize the speed and quality of identifications.     

Estimating brocket deer (<Emphasis Type="Italic">Mazama gouazoubira</Emphasis> and <Emphasis Type="Italic">M. americana</Emphasis>) abundance by dung pellet counts and other indices in seasonal Chiquitano forest habitats of Santa Cruz,Bolivia

We assessed dung and track counts for indexing brocket deer abundance in seasonal habitats on a ranch where hunting is not practiced in the Bolivian lowlands. Surveys were replicated along four 10-km trails (totaling 180 km in the wet season and 90 km in the dry season) through four semideciduous forest habitats. Dung pellets and tracks were identified as belonging to Mazama gouazoubira or M. americana by size and shape. Pellet groups lasted more than 1 month during the dry season, but decayed within 1–2 weeks in the wet season. Mean density estimates based on dung counts varied widely between seasons for gray brockets (from 6.77±4.0 to 30.57±23.5 ind/km²; mean±SD) but not for reds (from 3.52±4.6 to 6.98±7.2 ind/km²). These values were probably too high due to underestimation of daily deposition rate, and were reduced during the wet season because of dung decay. We found consistently more dung in the dry season and more tracks in the wet season. Sightings of red brockets were too few for line-transect analysis (n=6), but those of gray brockets (n=42) produced an overall estimate of 5.6 ind/km² (95% CI=3.5–9.0 ind/km²). Different estimates indicated that gray brockets were more abundant than reds in all situations, except perhaps in the riverine forest. Environmental factors affected these indices of abundance differently, and while we recommend the use of dung counts in dry-season scenarios, we think that index reliability should be assessed locally before conducting population comparisons.     

The Discovery,Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.     

Infection of Seedlings of Alfalfa and Red Clover by Concomitant Populations of Meloidogyne incognita and Pratylenchus penetrans

Invasion of 2-day-old seedlings of ''Buffalo'' alfalfa and ''Kenland'' red clover by larvae of M. incognita and adults of P. penetrans, during 1-3 day periods of incubation at 24 C, was investigated in 50-mm petri dishes on 1% agar. Penetration by both nematodes increased arithmetically with increased numbers in inocula. P. penetrans invaded alfalfa more readily than red cover, but M. incognita invaded red clover more readily than alfalfa. Both nematodes inhibited root-elongation of alfalfa more than that of red clover. In combinations of 10 and 50 of both nematodes, invasion of both plants by both nematodes was the same as for each nematode alone. Penetration by M. incognita into alfalfa, but not into red clover, was significantly reduced when combinations of 50 M. incognita and 200 P. penetrans were inoculated simultaneously. In the presence of large numbers of entrant P. penetrans in both plants, penetration by M. incognita was highly significantly reduced. Penetration by P. penetrans was unaffected in the reciprocal situations.     

Temporal patterns of visitation of birds and mammals at mineral licks in the Peruvian Amazon

Mineral licks are key ecological resources for many species of birds and mammals in Amazonia, providing essential dietary nutrients and clays, yet little is known about which species visit and their behaviors at the mineral licks. Studying visitation and behavior at mineral licks can provide insight into the lives of otherwise secretive and elusive species. We assessed which species visited mineral licks, when they visited, and whether visits and the probability of recording groups at mineral licks were seasonal or related to the lunar cycle. We camera trapped at 52 mineral licks in the northeastern Peruvian Amazon and detected 20 mammal and 13 bird species over 6,255 camera nights. Generalized linear models assessed visitation patterns and records of groups in association with seasonality and the lunar cycle. We report nocturnal curassows (Nothocrax urumutum) visiting mineral licks for the first time. We found seasonal trends in visitation for the black agouti (Dasyprocta fuliginosa), red howler monkey (Alouatta seniculus), blue‐throated piping guan (Pipile cumanensis), red brocket deer (Mazama americana), collared peccary (Pecari tajacu), and tapir (Tapirus terrestris). Lunar trends in visitation occurred for the paca (Cuniculus paca), Brazilian porcupine (Coendou prehensilis), and red brocket deer. The probability of recording groups (>1 individual) at mineral licks was seasonal and related to lunar brightness for tapir. Overall, our results provide important context for how elusive species of birds and mammals interact with these key ecological resources on a landscape scale. The ecological importance of mineral licks for these species can provide context to seasonal changes in species occupancy and movement.

Many species of animals in the Amazon visit mineral licks, consuming soil to supplement their diet. Using camera trap data, we show that visits to mineral licks are seasonal for many species. These trends in visitation are likely due to species‐specific factors such as reproduction, predator avoidance, seasonal diet shifts, and resource use.     

Role of Nematodes,Nematicides, and Crop Rotation on the Productivity and Quality of Potato,Sweet Potato,Peanut, and Grain Sorghum

The objective of this experiment was to determine the effects of fenamiphos 15G and short-cycle potato (PO)-sweet potato (SP) grown continuously and in rotation with peanut (PE)-grain sorghum (GS) on yield, crop quality, and mixed nematode population densities of Meloidogyne arenaria, M. hapla, M. incognita, and Mesocriconema ornatum. Greater root-gall indices and damage by M. hapla and M. incognita occurred on potato than other crops. Most crop yields were higher and root-gall indices lower from fenamiphos-treated plots than untreated plots. The total yield of potato in the PO-SP and PO-SP-PE-GS sequences increased from 1983 to 1985 in plots infested with M. hapla or M. arenaria and M. incognita in combination and decreased in 1986 to 1987 when root-knot nematode populations shifted to M. incognita. The total yields of sweet potato in the PO-SP-PE-GS sequence were similar in 1983 and 1985, and declined each year in the PO-SP sequence as a consequence of M. incognita population density increase in the soil. Yield of peanut from soil infested with M. hapla increased 82% in fenamiphos-treated plots compared to untreated plots. Fenamiphos treatment increased yield of grain sorghum from 5% to 45% over untreated controls. The declining yields of potato and sweet potato observed with both the PO-SP and PO-SP-PE-GS sequences indicate that these crop systems should not be used longer than 3 years in soil infested with M. incognita, M. arenaria, or M. hapla. Under these conditions, these two cropping systems promote a population shift in favor of M. incognita, which is more damaging to potato and sweet potato than M. arenaria and M. hapla.