Parasite dynamics in North American monarchs predicted by host density and seasonal migratory culling.

Insect-pathogen dynamics can show seasonal and inter-annual variations that covary with fluctuations in insect abundance and climate. Long-term analyses are especially needed to track parasite dynamics in migratory insects, in part because their vast habitat ranges and high mobility might dampen local effects of density and climate on infection prevalence. Monarch butterflies Danaus plexippus are commonly infected with the protozoan Ophryocystis elektroscirrha (OE). Because this parasite lowers monarch survival and flight performance, and because migratory monarchs have experienced declines in recent decades, it is important to understand the patterns and drivers of infection. Here we compiled data on OE infection spanning 50 years, from wild monarchs sampled in the United States, Canada and Mexico during summer breeding, fall migrating and overwintering periods. We examined eastern versus western North American monarchs separately, to ask how abundance estimates, resource availability, climate and breeding season length impact infection trends. We further assessed the intensity of migratory culling, which occurs when infected individuals are removed from the population during migration. Average infection prevalence was four times higher in western compared to eastern subpopulations. In eastern North America, the proportion of infected monarchs increased threefold since the mid-2000s. In the western region, the proportion of infected monarchs declined sharply from 2000 to 2015, and increased thereafter. For both eastern and western subpopulations, years with greater summer adult abundance predicted greater infection prevalence, indicating that transmission increases with host breeding density. Environmental variables (temperature and NDVI) were not associated with changes in the proportion of infected adults. We found evidence for migratory culling of infected butterflies, based on declines in parasitism during fall migration. We estimated that tens of millions fewer monarchs reach overwintering sites in Mexico as a result of OE, highlighting the need to consider the parasite as a potential threat to the monarch population. Increases in infection among eastern North American monarchs post-2002 suggest that changes to the host's ecology or environment have intensified parasite transmission. Further work is needed to examine the degree to which human practices, such as mass caterpillar rearing and the widespread planting of exotic milkweed, have contributed to this trend.

Ophryocystis sitonae sp. nov., (Neogregarinida: Ophryocystidae) parasitizing Sitona humeralis Stephens, 1831 (Coleoptera: Curculionidae).

Sitona humeralis Stephens 1831 (Coleoptera: Curculionidae) is an important pest of the Medicago and Vicia species in Turkey, and this study was conducted the determine the natural pathogens of this beetle. In the present study, a new neogregarine was observed in Malpighian tubules of the S. humeralis, collected from Ordu (Turkey) on the wild Medicago species. The yellowish oocysts were the most notable feature of the current neogregarine. The Giemsa-stained mature oocysts were fusiform shaped and measured 8.7 ± 0.7 (7.12-11.11) µm in length and 4.1 ± 0.3 (3.05-5.01) µm in width. The smooth oocyst wall was relatively thin (175-230 nm), and polar plugs were non-evident (weight = 380 nm, height = 500 nm). The 18S rDNA gene of the current neogregarine was sequenced and compared with fifteen sequences from GenBank. Morphological, ultrastructural, and molecular features indicate that the described neogregarine in S. humeralis differed from the all known Ophryocystis species and named here Ophryocystis sitonae sp. nov.

Comparison between Apicystis cryptica sp. n. and Apicystis bombi (Arthrogregarida, Apicomplexa): Gregarine parasites that cause fat body hypertrophism in bees.

The molecular divergence, morphology and pathology of a cryptic gregarine that is related to the bee parasite Apicystis bombi Lipa and Triggiani, 1996 is described. The 18S ribosomal DNA gene sequence of the new gregarine was equally dissimilar to that of A. bombi and the closest related genus Mattesia Naville, 1930, although phylogenetic analysis supported a closer relation to A. bombi. Pronounced divergence with A. bombi was found in the ITS1 sequence (69.6% similarity) and seven protein-coding genes (nucleotide 78.05% and protein 90.2% similarity). The new gregarine was isolated from a Bombus pascuorum Scopoli, 1763 female and caused heavy hypertrophism of the fat body tissue in its host. In addition, infected cells of the hypopharyngeal gland tissue, an important excretory organ of the host, were observed. Mature oocysts were navicular in shape and contained four sporozoites, similar to A. bombi oocysts. Given these characteristics, we proposed the name Apicystis cryptica sp. n. Detections so far indicated that distribution and host species occupation of Apicystis spp. overlap at least in Europe, and that historical detections could not discriminate between them. Specific molecular assays were developed that can be implemented in future pathogen screens that aim to discriminate Apicystis spp. in bees.

Ophryocystis anatoliensis sp. nov., a new neogregarine pathogen of the chrysomelid beetle Chrysomela populi.

Chrysomela populi (Coleoptera: Chrysomelidae) is the most abundant and most important pest species that causes damage to poplar trees. Members of the family Chrysomelidae are frequently infected by protist pathogens but no neogregarine has been reported to date at the species level. In the present study we identify a new neogregarine pathogen from the chrysomelid C. populi. The infection was observed in the Malpighian tubules of adult beetles. A reddening of the Malpighian tubules was the most distinctive symptom of the infection. Single fusiform oocysts (9.8×4.7µm) were formed within a gamontocyst. The polar plugs were very thin, varying from 380 to 525nm in thickness. The oocyst wall was smooth and also quite thin (90-120nm). Morphological and ultrastructural characteristics of the pathogen indicate that the described neogregarine in C. populi is clearly different from known Ophryocystis species which infect coleopterans. Therefore, the neogregarine pathogen was determined to be a newly discovered species and named Ophryocystis anatoliensis sp. nov.

Aranciocystis muskarensis n. gen., n. sp., a neogregarine pathogen of the Anisoplia segetum Herbst (Coleoptera: Scarabaeidae).

In this study, a new genus and species of neogregarine which is a pathogen of Anisoplia segetum Herbst (Coleoptera: Scarabaeidae), is described. The adult beetles of A. segetum were collected from Nevsehir, Turkey and neogregarine infection rates were determined as 18.52%. The Giemsa-stained mature oocysts are lemon-shaped and measured 9.34±0.82µm in length and 5.77±0.77µm in width. The oocyst wall surface of the mature oocysts is similar to an osage orange (tuberculate). Morphological, ultrastructural and molecular features indicate that the previously undescribed neogregarine is dissimilar to all known neogregarine taxa and represents the first record from Anisoplia segetum and is named here as Aranciocystis muskarensis n. gen., n. sp.

The effects of single and mixed infections of Apicystis bombi and deformed wing virus in Bombus terrestris.

Many pollinators are currently suffering from declines, diminishing their gene pool and increasing their vulnerability to parasites. Recently, an increasing diversity of parasites has been recorded in bumblebees, yet for many, knowledge of their virulence and hence the risk their presence poses, is lacking. The deformed wing virus (DWV), known to be ubiquitous in honey bees, has now been detected in bumblebees. In addition, the neogregarine Apicystis bombi has been discovered to be more prevalent than previously thought. Here, we assess for the first time the lethal and sublethal effects of these parasites during single and mixed infections of worker bumblebees (Bombus terrestris). Fifteen days after experimental exposure, 22% of bees exposed to A. bombi, 50% of bees exposed to DWV and 86% of bees exposed to both parasites had died. Bumblebees that had ingested A. bombi had increased sucrose sensitivity (SS) and a lower lipid:body size ratio than control bees. While dual infected bumblebees showed no increase in SS. Overall, we find that A. bombi exhibits both lethal and sublethal effects. DWV causes lethal effect and may reduce the sub lethal effects imposed by A. bombi. The results show that both parasites have significant, negative effects on bumblebee health, making them potentially of conservation concern.

Loss of migratory behaviour increases infection risk for a butterfly host.

Long-distance animal migrations have important consequences for infectious disease dynamics. In some cases, migration lowers pathogen transmission by removing infected individuals during strenuous journeys and allowing animals to periodically escape contaminated habitats. Human activities are now causing some migratory animals to travel shorter distances or form sedentary (non-migratory) populations. We focused on North American monarch butterflies and a specialist protozoan parasite to investigate how the loss of migratory behaviours affects pathogen spread and evolution. Each autumn, monarchs migrate from breeding grounds in the eastern US and Canada to wintering sites in central Mexico. However, some monarchs have become non-migratory and breed year-round on exotic milkweed in the southern US. We used field sampling, citizen science data and experimental inoculations to quantify infection prevalence and parasite virulence among migratory and sedentary populations. Infection prevalence was markedly higher among sedentary monarchs compared with migratory monarchs, indicating that diminished migration increases infection risk. Virulence differed among parasite strains but was similar between migratory and sedentary populations, potentially owing to high gene flow or insufficient time for evolutionary divergence. More broadly, our findings suggest that human activities that alter animal migrations can influence pathogen dynamics, with implications for wildlife conservation and future disease risks.

Genetic Factors and Host Traits Predict Spore Morphology for a Butterfly Pathogen.

Monarch butterflies (Danaus plexippus) throughout the world are commonly infected by the specialist pathogen Ophryocystis elektroscirrha (OE). This protozoan is transmitted when larvae ingest infectious stages (spores) scattered onto host plant leaves by infected adults. Parasites replicate internally during larval and pupal stages, and adult monarchs emerge covered with millions of dormant spores on the outsides of their bodies. Across multiple monarch populations, OE varies in prevalence and virulence. Here, we examined geographic and genetic variation in OE spore morphology using clonal parasite lineages derived from each of four host populations (eastern and western North America, South Florida and Hawaii). Spores were harvested from experimentally inoculated, captive-reared adult monarchs. Using light microscopy and digital image analysis, we measured the size, shape and color of 30 replicate spores per host. Analyses examined predictors of spore morphology, including parasite source population and clone, parasite load, and the following host traits: family line, sex, wing area, and wing color (orange and black pigmentation). Results showed significant differences in spore size and shape among parasite clones, suggesting genetic determinants of morphological variation. Spore size also increased with monarch wing size, and monarchs with larger and darker orange wings tended to have darker colored spores, consistent with the idea that parasite development depends on variation in host quality and resources. We found no evidence for effects of source population on variation in spore morphology. Collectively, these results provide support for heritable variation in spore morphology and a role for host traits in affecting parasite development.