Prior to 1965, Singapore was part of the Malaya (now Malaysia) and was usually not mentioned when mosquito records were reported for Malaya. Consequently, many species that occurred in Singapore were not listed in the world mosquito catalog, and the available checklist for Singapore since 1986 is incomplete, with some imprecise species information. In updating this checklist, we examined and verified mosquito specimens collected from Singapore in various depositories, including a thorough review of past taxonomic literature. Here, we report a checklist of 182 mosquito species, 33 new distribution records, and a consolidated status list of vectors for Singapore. As Singapore is a travel hub and hosts one of the busiest container ports in the world, there is a risk of introducing mosquito species and their associated pathogens of human disease to the country. Hence, the distribution records are important to increase our knowledge on mosquito ecology as well as to understand the risk of newly introduced vectors and their associated pathogens.

Background Wolbachia are intracellular bacterial endosymbionts found in most insect lineages. In mosquitoes, the influence of these endosymbionts on host reproduction and arboviral transmission has spurred numerous studies aimed at using Wolbachia infection as a vector control technique. However, there are several knowledge gaps in the literature and little is known about natural Wolbachia infection across species, their transmission modes, or associations between various Wolbachia lineages and their hosts. This study aims to address these gaps by exploring mosquito-Wolbachia associations and their evolutionary implications. Methods We conducted tissue-specific polymerase chain reaction screening for Wolbachia infection in the leg, gut and reproductive tissues of wild mosquitoes from Singapore using the Wolbachia surface protein gene (wsp) molecular marker. Mosquito-Wolbachia associations were explored using three methods—tanglegram, distance-based, and event-based methods—and by inferred instances of vertical transmission and host shifts. Results Adult mosquitoes (271 specimens) representing 14 genera and 40 species were screened for Wolbachia. Overall, 21 species (51.2%) were found positive for Wolbachia, including five in the genus Aedes and five in the genus Culex. To our knowledge, Wolbachia infections have not been previously reported in seven of these 21 species: Aedes nr. fumidus, Aedes annandalei, Uranotaenia obscura, Uranotaenia trilineata, Verrallina butleri, Verrallina sp. and Zeugnomyia gracilis. Wolbachia were predominantly detected in the reproductive tissues, which is an indication of vertical transmission. However, Wolbachia infection rates varied widely within a mosquito host species. There was no clear signal of cophylogeny between the mosquito hosts and the 12 putative Wolbachia strains observed in this study. Host shift events were also observed. Conclusions Our results suggest that the mosquito-Wolbachia relationship is complex and that combinations of transmission modes and multiple evolutionary events likely explain the observed distribution of Wolbachia diversity across mosquito hosts. These findings have implications for a better understanding of the diversity and ecology of Wolbachia and for their utility as biocontrol agents.

The domesticated house cricket, Acheta domesticus (Linnaeus, 1758), has been lauded as a more sustainable protein source than traditional livestock to meet the ever-increasing demand for food. There is also a potential of using waste food, such as okara (soybean residue from the manufacture of soy milk and tofu) and waste vegetables which are commonly available in many parts of Southeast Asia, to feed these insects. Food wastage can be reduced while increasing the food supply if the crickets can grow and survive well on waste food. Thus, we investigated the viability of rearing A. domesticus on oligidic diets, specifically waste vegetables and okara, to optimise the use of waste food as feed to the crickets. We monitored cricket mortality and fresh weight of 32 individually housed crickets subjected to different diet treatments, every other day for 45 days. The crickets reared on Brassica rapa (xiao bai cai; XBC) and okara mixture did not grow and survive as well as those reared on the XBC and dog food mixture, contradicting our initial prediction that okara is a suitable cricket feed, even though it has been considered one of the best oligidic diets. We also did not find any evidence that crickets reared on mixed diets of XBC and okara food grew or survived better than single diets of XBC or okara. This showed that okara cannot always replace premium animal feed to rear A. domesticus crickets.

1. A significant proportion of vector-borne diseases are transmitted by blood-sucking dipterans, including mosquitoes. Understanding transmission risks requires accurate identification of species across heterogeneous habitats, but many cryptic and polymorphic species are overlooked when using morphological identification. Estimates of mosquito diversity are typically based on adult female trapping methods which tend to target host-seeking species and may represent a biased snapshot of community structure. Unfortunately, diversity estimates based on larval data are rarely included in mosquito ecological analyses.2. We carried out adult and larval sampling over 6 months in Singapore using an integrative approach of morphological identification and molecular delineation with mini-barcodes (313 bp) generated on a Next-Generation Sequencing platform to obtain species estimates. We collected 3,201 mosquitoes across 58 species (14 genera). Notably, 16 species were collected only through larval sampling and 22 species were only resolved using mini-barcodes. Of the latter, we identified three morphologically similar species groups and documented several intraspecific polymorphisms.3. We compared adult-only data against a full dataset (adult + larval + mini-barcode).The species accumulation curves reached an asymptote for all but one site when using the latter and non-metric multidimensional scaling (NMDS) revealed that mosquito communities were only well separated when using the full dataset.Overall, the full dataset reflects a more defined and accurate community structure across all sites. We find that many mosquito species are niche-specific and several species were generally influenced by tree cover, rainfall and presence of large water bodies. Synthesis and applications.We report the first successful use of mini-barcodes on mosquitoes and demonstrate its utility in delineating multiple challenging species groups. We recommend the use of both morphological and molecular identification methods for ecological studies and vector surveillance. Misidentification in species estimation, especially for medically relevant insect groups, can lead to conflicting reports and slows down vector control efforts. We provide evidence

Tetrigidae are an ancient group of grasshoppers and, similar to many other insects, have associations and preferences for specific microhabitats and habitats. The ecology of pygmy grasshoppers in Southeast Asia is generally under studied, especially in threatened habitats such as freshwater swamp forests. A study in Nee Soon Swamp forest, Singapore, was conducted to investigate association of limno-terrestrial pygmy grasshoppers with waterbodies and microhabitat. Specifically, we looked at the abundance and species assemblage of all pygmy grasshoppers. We correlated the abundance with major gradients of variation summarizing substrate and vegetation types along belt transects where sampling was performed. We found that pygmy grasshoppers in general are associated with wetter microhabitat conditions rather than the main streams in the swamp forest (i.e., water bodies). This is despite differences in microhabitat conditions of belt transects nearer to and further away from the main streams. We also found that pygmy grasshopper abundance is associated with the wetness of dicot leaf litter. We inferred that the abundance of food resources and suitability for egg development may explain their preference for wet microhabitats. We also found that the same patterns applied for adults and juveniles, suggesting that there is no demographic difference or ontogenetic shift in microhabitat association. Lastly, the adult assemblage can also be correlated to microhabitats. Based on our findings, we propose that pygmy grasshoppers can also be suitable bioindicators for the freshwater swamp forest, owing to their sensitivity to microhabitat conditions.