1. During the 2009-2010 overwintering season and following a 15-year downward trend, the total area in Mexico occupied by the eastern North American population of overwintering monarch butterflies reached an all-time low. Despite an increase, it remained low in 2010-2011.2. Although the data set is small, the decline in abundance is statistically significant using both linear and exponential regression models.3. Three factors appear to have contributed to reduce monarch abundance: degradation of the forest in the overwintering areas; the loss of breeding habitat in the United States due to the expansion of GM herbicide-resistant crops, with consequent loss of milkweed host plants, as well as continued land development; and severe weather.4. This decline calls into question the long-term survival of the monarchs' migratory phenomenon.Resú men. 1. Durante la temporada invernal 2009-2010, y siguiendo una tendencia a la baja de 15 an˜os, la superficie total ocupada por mariposas monarca en Me´xico, provenientes del este Ame´rica del Norte, llego´a su punto ma´s bajo. A pesar de su incremento, dicha superficie siguio´siendo baja en 2010-2011.2. Aunque que el conjunto de datos disponibles es au´n pequen˜o, esta disminucio´n de la abundancia de mariposas es estadı´sticamente significativa, tanto si se usan modelos de regresio´n lineales como exponenciales.3. Hay tres factores que parecen haber contribuido con esta tendencia de reduc-cio´n del nu´mero de mariposas: la degradacio´n de bosque en las a´reas de invernacio´n en Me´xico; la pe´rdida de ha´bitat de reproduccio´n en los Estados Unidos, debido a la expansio´n de cultivos gene´ticamente modificados resistentes a herbicidas, con la consiguiente pe´rdida de las plantas hospederas de algodoncillo, y por continuos cambios en el uso del suelo no favorables para ellas; y, las recientes condiciones cli-ma´ticas severas.4. Esta disminucio´n hace que nos cuestionemos sobre la posibilidad de supervivencia a largo plazo del feno´meno migratorio de las mariposas monarca.

Wing membranes of laboratory and field-reared monarch butterflies (Danaus plexippus) were analyzed for their stable-hydrogen (δD) and carbon (δC) isotope ratios to determine whether this technique could be used to identify their natal origins. We hypothesized that the hydrogen isotopic composition of monarch butterfly wing keratin would reflect the hydrogen isotope patterns of rainfall in areas of natal origin where wings were formed. Monarchs were reared in the laboratory on milkweed plants (Asclepias sp.) grown with water of known deuterium content, and, with the assistance of volunteers, on native milkweeds throughout eastern North America. The results show that the stable hydrogen isotopic composition of monarch butterflies is highly correlated with the isotopic composition of the milkweed host plants, which in turn corresponds closely with the long-term geographic patterns of deuterium in rainfall. Stable-carbon isotope values in milkweed host plants were similarly correlated with those values in monarch butterflies and showed a general pattern of enrichment along a southwest to northeast gradient bisecting the Great Lakes. These findings indicate that natal origins of migratory and wintering monarchs in Mexico can be inferred from the combined δD and δC isotopic signatures in their wings. This relationship establishes that analysis of hydrogen and carbon isotopes can be used to answer questions concerning the biology of migratory monarch butterflies and provides a new approach to tracking similar migratory movements of other organisms.

The Eastern, migratory population of monarch butterflies (Danaus plexippus), an iconic North American insect, has declined by ~80% over the last decade. The monarch’s multi-generational migration between overwintering grounds in central Mexico and the summer breeding grounds in the northern U.S. and southern Canada is celebrated in all three countries and creates shared management responsibilities across North America. Here we present a novel Bayesian multivariate auto-regressive state-space model to assess quasi-extinction risk and aid in the establishment of a target population size for monarch conservation planning. We find that, given a range of plausible quasi-extinction thresholds, the population has a substantial probability of quasi-extinction, from 11–57% over 20 years, although uncertainty in these estimates is large. Exceptionally high population stochasticity, declining numbers, and a small current population size act in concert to drive this risk. An approximately 5-fold increase of the monarch population size (relative to the winter of 2014–15) is necessary to halve the current risk of quasi-extinction across all thresholds considered. Conserving the monarch migration thus requires active management to reverse population declines, and the establishment of an ambitious target population size goal to buffer against future environmentally driven variability.

The monarch butterfly (Danaus plexippus) population in North America has sharply declined over the last two decades. Despite rising concern over the monarch butterfly's status, no comprehensive study of the factors driving this decline has been conducted. Using partial least-squares regressions and time-series analysis, we investigated climatic and habitat-related factors influencing monarch population size from 1993 to 2014. Potential threats included climatic factors, habitat loss (milkweed and overwinter forest), disease and agricultural insecticide use (neonicotinoids). While climatic factors, principally breeding season temperature, were important determinants of annual variation in abundance, our results indicated strong negative relationships between population size and habitat loss variables, principally glyphosate use, but also weaker negative effects from the loss of overwinter forest and breeding season use of neonicotinoids. Further declines in population size because of glyphosate application are not expected. Thus, if remaining threats to habitat are mitigated we expect climate-induced stochastic variation of the eastern migratory population of monarch butterfly around a relatively stationary population size.

The eastern migratory population of monarch butterflies (Danaus plexippus plexippus) has declined by >80% within the last two decades. One possible cause of this decline is the loss of ≥1.3 billion stems of milkweed (Asclepias spp.), which monarchs require for reproduction. In an effort to restore monarchs to a population goal established by the US Fish and Wildlife Service and adopted by Mexico, Canada, and the US, we developed scenarios for amending the Midwestern US landscape with milkweed. Scenarios for milkweed restoration were developed for protected area grasslands, Conservation Reserve Program land, powerline, rail and roadside rights of way, urban/suburban lands, and land in agricultural production. Agricultural land was further OPEN ACCESS RECEIVED

Helianthella quinquenervis (Asteraceae), the aspen sunflower, secretes sugar and amino acid rich nectar from involucral bracts during bud and flowering stages. The nectar is usually collected by ants as fast as it is secreted. H. quinquenervis is subject to predispersal seed predation by larvae of several insect species, primarily tephritid flies, an agromyzid fly, and 3 Lepidoptera. When present, ants interrupt oviposition efforts by the female flies but do not appear to be effective against lepidopteran or mammalian herbivores. The degree of protection by ants conferred upon plants was investigated by excluding ants from some plants and by correlating ant density with subsequent damage to ovules and developing seeds. At higher elevations (2896 and 3091 m) damage to plants with ants was significantly reduced compared with plants without ants. At a lower elevation (2734 m) where seed predator and ant densities were highest, the ants were less effective as deterrents and predation on ovules and seeds usually exceeded 60%. Flowers of some other composite species in the same habitats appear to be more effectively protected from predation by chemical deterrents, raising questions of the relative benefits of chemical deterrents compared to ants as a means of protection.

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes.

The composition of the five-component honey bee queen mandibular gland pheromone (QMP) of mated European honey bee queens was compared to those of virgin and drone-laying (i.e., laying only haploid unfertilized eggs that develop into males), European queens and Africanized mated queens. QMP of mated European queens showed significantly greater quantities of individual components than all queen types compared, except for a significantly greater quantity of 9-hydroxy-(E)-2-decenoic acid (9-HDA) found in Africanized queens. Glands of European drone-laying queens contained quantities intermediate between virgin and mated queens, reflecting their intermediate reproductive state and age. QMP ontogeny shifts from a high proportion of 9-keto-(E)-2-decenoic acid (ODA) in young unmated queens to roughly equal proportions of ODA and 9-HDA in mated queens. A biosynthetic shift occurs after mating that results in a greater proportion of 9-HDA, methylp-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) production, accompanied by a decreased proportion of ODA. Africanized QMP proportions of ODA and 9-HDA were significantly different from European queens. A quantitative definition of a "queen equivalent" of QMP is proposed for the various queen types, and a standard queen equivalent for mated European honeybee queen mandibular gland pheromone is adopted as 200µg ODA, 80µg 9-HDA, 20µg HOB, and 2 µg HVA.