Dynamics of stored lipids in fall migratory monarch butterflies (Danaus plexippus): Nectaring in northern Mexico allows recovery from droughts at higher latitudes.

The eastern population of the North American monarch butterfly (Danaus plexippus) overwinters from November through March in the high-altitude (3000 m+) forests of central Mexico during which time they rely largely on stored lipids. These are acquired during larval development and the conversion of sugars from floral nectar by adults. We sampled fall migrant monarchs from southern Canada through the migratory route to two overwintering sites in 2019 (n = 10 locations), 2020 (n = 8 locations) and 2021 (n = 7 locations). Moderate to extreme droughts along the migratory route were expected to result in low lipid levels in overwintering monarchs but our analysis of lipid levels of monarchs collected at overwintering sites indicated that in all years most had high levels of lipids prior to winter. Clearly, a significant proportion of lipids were consistently acquired in Mexico during the last portion of the migration. Drought conditions in Oklahoma, Texas and northern Mexico in 2019 resulted in the lowest levels of lipid mass and wing loading observed in that year but with higher levels at locations southward in Mexico to the overwintering sites. Compared with 2019, lipid levels increased during the 2020 and 2021 fall migrations but were again higher during the Mexican portion of the migration than for Oklahoma and Texas samples, emphasizing a recovery of lipids as monarchs advanced toward the overwintering locations. In all 3 years, body water was highest during the Canada-USA phase of migration but then declined during the nectar foraging phase in Mexico before recovering again at the overwintering sites. The increase in mass and lipids from those in Texas to the overwintering sites in Mexico indicates that nectar availability in Mexico can compensate for poor conditions experienced further north. Our work emphasizes the need to maintain the floral and therefore nectar resources that fuel both the migration and storage of lipids throughout the entire migratory route.

The cost of movement: assessing energy expenditure in a long-distant ectothermic migrant under climate change.

Migration is an energetically taxing phenomenon as animals move across vast, heterogeneous landscapes where the cost of transport is impacted by permissible ambient conditions. In this study, we assessed the energetic demands of long-distance migration in a multigenerational ectothermic migrant, the monarch butterfly (Danaus plexippus). We tested the hypotheses that temperature-dependent physiological processes reduce energy reserves faster during migration than previously estimated, and that increasing climatic temperatures resulting from the climate crisis will intensify baseline daily energy expenditure. First, we reared monarchs under laboratory conditions to assess energy and mass conversion from fifth instar to adult stages, as a baseline for migratory adult mass and ontogenetic shifts in metabolic rate from larvae to adult. Then, using historical tag-recapture data, we estimated the movement propensity and migratory pace of autumn migrants using computer simulations and subsequently calculated energy expenditure. Finally, we estimated the energy use of monarchs based on these tag-recapture data and used this information to estimate daily energy expenditure over a 57 year period. We found support for our two hypotheses, noting that incorporating standard metabolic rate into estimates of migratory energy expenditure shows higher energy demand and that daily energy expenditure has been gradually increasing over time since 1961. Our study shows the deleterious energetic consequences under current climate change trajectories and highlights the importance of incorporating energetic estimates for understanding migration by small, ectothermic migrants.

Monarch butterfly population decline in North America: identifying the threatening processes.

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.

Interpreting surveys to estimate the size of the monarch butterfly population: Pitfalls and prospects.

To assess the change in the size of the eastern North American monarch butterfly summer population, studies have used long-term data sets of counts of adult butterflies or eggs per milkweed stem. Despite the observed decline in the monarch population as measured at overwintering sites in Mexico, these studies found no decline in summer counts in the Midwest, the core of the summer breeding range, leading to a suggestion that the cause of the monarch population decline is not the loss of Midwest agricultural milkweeds but increased mortality during the fall migration. Using these counts to estimate population size, however, does not account for the shift of monarch activity from agricultural fields to non-agricultural sites over the past 20 years, as a result of the loss of agricultural milkweeds due to the near-ubiquitous use of glyphosate herbicides. We present the counter-hypotheses that the proportion of the monarch population present in non-agricultural habitats, where counts are made, has increased and that counts reflect both population size and the proportion of the population observed. We use data on the historical change in the proportion of milkweeds, and thus monarch activity, in agricultural fields and non-agricultural habitats to show why using counts can produce misleading conclusions about population size. We then separate out the shifting proportion effect from the counts to estimate the population size and show that these corrected summer monarch counts show a decline over time and are correlated with the size of the overwintering population. In addition, we present evidence against the hypothesis of increased mortality during migration. The milkweed limitation hypothesis for monarch decline remains supported and conservation efforts focusing on adding milkweeds to the landscape in the summer breeding region have a sound scientific basis.

Density estimates of monarch butterflies overwintering in central Mexico.

Given the rapid population decline and recent petition for listing of the monarch butterfly (Danaus plexippus L.) under the Endangered Species Act, an accurate estimate of the Eastern, migratory population size is needed. Because of difficulty in counting individual monarchs, the number of hectares occupied by monarchs in the overwintering area is commonly used as a proxy for population size, which is then multiplied by the density of individuals per hectare to estimate population size. There is, however, considerable variation in published estimates of overwintering density, ranging from 6.9-60.9 million ha-1. We develop a probability distribution for overwinter density of monarch butterflies from six published density estimates. The mean density among the mixture of the six published estimates was ∼27.9 million butterflies ha-1 (95% CI [2.4-80.7] million ha-1); the mixture distribution is approximately log-normal, and as such is better represented by the median (21.1 million butterflies ha-1). Based upon assumptions regarding the number of milkweed needed to support monarchs, the amount of milkweed (Asclepias spp.) lost (0.86 billion stems) in the northern US plus the amount of milkweed remaining (1.34 billion stems), we estimate >1.8 billion stems is needed to return monarchs to an average population size of 6 ha. Considerable uncertainty exists in this required amount of milkweed because of the considerable uncertainty occurring in overwinter density estimates. Nevertheless, the estimate is on the same order as other published estimates. The studies included in our synthesis differ substantially by year, location, method, and measures of precision. A better understanding of the factors influencing overwintering density across space and time would be valuable for increasing the precision of conservation recommendations.

Quasi-extinction risk and population targets for the Eastern, migratory population of monarch butterflies (Danaus plexippus).

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.

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases.

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 flight physiology of reproductives of Africanized, European, and hybrid honeybees (Apis mellifera).

Neotropical African honeybees (Apis mellifera scutellata), in the process of spreading throughout tropical and subtropical regions of the Americas, hybridize with and mostly replace European honeybees (primarily Apis mellifera mellifera and Apis mellifera ligustica). To help understand this process, we studied the effect of lineage (African, European, or hybrid) on the flight physiology of honeybee reproductives. Flight metabolic rates were higher in queens and drones of African lineage than in European or hybrid bees, as has been previously found for foraging workers. These differences were associated with higher thorax/body mass ratios and higher thorax-specific metabolic rates in African lineage bees. Queens were reared in common colonies, so these metabolic and morphological differences are likely to be genetic in origin. African drones had higher wing beat frequencies and thorax temperatures than European or hybrid bees. Hybrids were intermediate for many parameters, but hybrid queen mass-specific flight metabolic rates were low relative to Africans and were nonlinearly affected by the proportion of African lineage, consistent with some negative heterosis for this trait.

Stable isotopes (δD and δ13C) are geographic indicators of natal origins of monarch butterflies in eastern North America.

Wing membranes of laboratory and field-reared monarch butterflies (Danaus plexippus) were analyzed for their stable-hydrogen (δD) and carbon (δ13C) 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 δ13C 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.

Demography and life history characteristics of two honey bee races (Apis mellifera).

Intra-colony demography and life history characteristics of neotropical Africanized and temperate European honey bearaces were compared under simulated feral conditions. Major differences in colony demography were found which nevertheless resulted in some similar reproductive characteristics. European colonies were larger than Africanized colonies, had more rapid initral growth rates of worker populations, showed better survivorship of brood and adult workers, and differed in patterns of worker age distribution. However, both races were similar in the brood and adult populations when colonies swarmed, the frequency and timing of swarming, and the number of workers in prime swarms. The factors most important in determining these colony growth and reproductive patterns were likely worker mortality rates, climate, and resource availability.