Using multi-marker DNA metabarcoding to reveal the diet of a scarce woodland bird

Understanding the role diet plays in the structure of food webs is vital, and dietary knowledge is key for conservation management success. There is limited knowledge of the diets of woodland bird species, due largely to difficulties in accurately identifying plant and invertebrate taxa being consumed. Here, we show the effectiveness of multi-marker faecal metabarcoding to provide the most in-depth dietary analysis of a generalist passerine, the Hawfinch (Coccothraustes coccothraustes, Linnaeus), to date. Faecal samples were obtained from 2016-2019 from Hawfinch populations prior to and during the breeding season throughout the UK. DNA was extracted from 263 samples and amplified using Internal Transcribed Spacer 2 (ITS2) and cytochrome C oxidase subunit I (COI) barcodes. Using high-throughput sequencing (HTS), we identified 49 and 97 ITS2 and COI zero radius operational taxonomic units (zOTUs) respectively which equated to reputed dietary items. The herbivorous element of Hawfinch diet was dominated by naturally occurring taxa such as beech (Fagus sylvatica, Linnaeus), hornbeam (Carpinus betulus, Linnaeus) and oak (Quercus sp., Linnaeus). The most taxon rich and commonly recorded invertebrate taxon identified was Lepidoptera. We found Hawfinch diet varied spatially, as well as between sexes. Hawfinch showed broad dietary plasticity and utilised multiple resources within their foraging environments. Our study shows the potential of multi-marker DNA metabarcoding to reveal subtle dietary differences, but also highlights the challenges of studying omnivorous species using metabarcoding methods.

Persistent mosquito fogging can be detrimental to non-target invertebrates in an urban tropical forest

Background Human population growth has led to biodiversity declines in tropical cities. While habitat loss and fragmentation have been the main drivers of urban biodiversity loss, man-made interventions to reduce health risks have also emerged as an unintentional threat. For instance, insecticide fogging to control mosquito populations has become the most common method of preventing the expansion of mosquito-borne diseases such as Dengue. However, the effectiveness of fogging in killing mosquitoes has been called into question. One concern is the unintended effect of insecticide fogging on non-target invertebrates that are crucial for the maintenance of urban ecosystems. Here, we investigate the impacts of fogging on: (1) target invertebrate taxon (Diptera, including mosquitoes); (2) non-target invertebrate taxa; and (3) the foraging behavior of an invertebrate pollinator taxon (Lepidoptera) within an urban tropical forest. Methods We carried out fogging with Pyrethroid insecticide (Detral 2.5 EC) at 10 different sites in a forest situated in the state of Selangor, Peninsular Malaysia. Across the sites, we counted the numbers of knocked-down invertebrates and identified them based on morphology to different taxa. We constructed Bayesian hierarchical Poisson regression models to investigate the effects of fogging on: (1) a target invertebrate taxon (Diptera) 3-h post-fogging; (2) selected non-target invertebrate taxa 3-h post-fogging; and (3) an invertebrate pollinator taxon (Lepidoptera) 24-h post-fogging. Results A total of 1,874 invertebrates from 19 invertebrate orders were knocked down by the fogging treatment across the 10 sites. Furthermore, 72.7% of the invertebrates counted 3-h post-fogging was considered dead. Our regression models showed that given the data and prior information, the probability that fogging had a negative effect on invertebrate taxa 3-h post-fogging was 100%, with reductions to 11% of the pre-fogging count of live individuals for the target invertebrate taxon (Diptera), and between 5% and 58% of the pre-fogging count of live individuals for non-target invertebrate taxa. For the invertebrate pollinator, the probability that fogging had a negative effect 24-h post-fogging was also 100%, with reductions to 53% of the pre-fogging count of live individuals. Discussion Our Bayesian models unequivocally demonstrate that fogging has detrimental effects on one pollinator order and non-target invertebrate orders, especially taxa that have comparatively lower levels of chitinisation. While fogging is effective in killing the target order (Diptera), no mosquitos were found dead in our experiment. In order to maintain urban biodiversity, we recommend that health authorities and the private sector move away from persistent insecticide fogging and to explore alternative measures to control adult mosquito populations.

The genetic structure of the marine flatworm Stylochoplana pusilla (Rhabditophora: Polycladida) and its use of intertidal snails

Although marine phylogeographers have accumulated knowledge of the evolutionary history of various invertebrates, there is a large bias among the taxa regarding genetic data. The order Polycladida is a typical example for which little genetic information at population level is available. Here, we focused on the polyclad flatworm Stylochoplana pusilla, distributed in the Japanese Pacific coastal area. Stylochoplana pusilla is known to have commensal relationships with certain intertidal snails, using snails (mainly Monodonta confusa) as a refugee house. During low tide, S. pusilla hides in the mantle cavity of snails to protect themselves from desiccation and predation. Here, we investigated the genetic structure of S. pusilla using a mitochondrial Cytochrome Oxidase I marker and the species diversity of snails used by it. We found that S. pusilla has high genetic diversity of its populations. While S. pusilla showed a significant genetic differentiation among populations, it was relatively low. In addition, we also showed that S. pusilla used several intertidal snail species which inhabit various coastal environments. The present study suggests S. pusilla has sufficient dispersal ability to connect among its local populations. Also, the range of available snails for S. pusilla may help the connectivity among local populations. We provide important knowledge about this invertebrate taxon with a unique ecology, which has been insufficiently studied.

The response of invertebrate populations in three undisturbed soils in southwestern Ontario, Canada, to variations in local soil properties, seasonal changes, and climate

Three distinctly different undisturbed mature forested sites at the northern limits of the Carolinian forest system in Lambton County, Ontario, were examined to test the hypothesis that the abundance of each order of soil invertebrates captured is dependent on a unique set of soil properties, seasonal changes, and climate variations. Sixteen independent variables were recorded over five consecutive years. With the exception of rainfall, air temperature, and soil temperature, means of the measured variables differed significantly (P < 0.05) among soils. Twenty-eight taxa of invertebrates were captured, of which Acari, Collembola, and Nematoda were most abundant. Only the mean of total abundance and the mean abundance of Acari, Nematoda, and Haplotaxida differed significantly (P < 0.05) among soils. Haplotaxida was the only taxon in all three soils found to be influenced significantly (P < 0.05) by seasonal variation. The usual mid-summer minimum in the abundance of Haplotaxida was latest and most clearly defined in the clay soil and earliest and least clearly defined in the sand soil. Regression analysis showed that each site is sufficiently separated in the factor space observed that the abundance of each invertebrate taxon is dependent on different combinations of local variables. The hypothesis was rejected.