Optimization of the feeding rate of Anopheles farauti s.s. colony mosquitoes in direct membrane feeding assays

Background: Direct membrane feeding assays (DMFA) are an important tool to study parasite transmission to mosquitoes. Mosquito feeding rates in these artificial systems require optimization, as there are a number of factors that potentially influence the feeding rates and there are no standardized methods that apply to all anopheline species. Methods: A range of parameters prior to and during direct membrane feeding (DMF) were evaluated for their impact on Anopheles farauti sensu stricto feeding rates, including the starving conditions and duration of starving prior to feeding, membrane type, DMF exposure time, mosquito age, feeding in the light versus the dark, blood volume, mosquito density and temperature of water bath. Results: The average successful DMFA feeding rate for An. farauti s.s. colony mosquitoes increased from 50 to 85% when assay parameters were varied. Overnight starvation and Baudruche membrane yielded the highest feeding rates but rates were also affected by blood volume in the feeder and the mosquito density in the feeding cups. Availability of water during the pre-feed starvation period did not significantly impact feeding rates, nor did the exposure duration to blood in membrane feeders, the age of mosquitoes (3, 5 and 7 days post-emergence), feeding in the light versus the dark, or the temperature (34 °C, 38 °C, 42 °C and 46 °C) of the water bath. Conclusion: Optimal feeding conditions in An. farauti s.s. DMFA were to offer 50 female mosquitoes in a cup (with a total surface area of ~ 340 cm2 with 1 mosquito/6.8 cm2) that were starved overnight 350–500 µL of blood (collected in heparin-coated Vacutainer tubes) per feeder in feeders with a surface area ~ 5 cm2 (with a maximum capacity of 1.5 mL of blood) via a Baudruche membrane, for at least 10–20 min

Insecticide resistance status of Aedes aegypti and Aedes albopictus mosquitoes in Papua New Guinea

Background: Aedes aegypti and Ae. albopictus are important vectors of infectious diseases, especially those caused by arboviruses such as dengue, chikungunya and Zika. Aedes aegypti is very well adapted to urban environments, whereas Ae. albopictus inhabits more rural settings. Pyrethroid resistance is widespread in these vectors, but limited data exist from the Southwest Pacific Region, especially from Melanesia. While Aedes vector ecology is well documented in Australia, where incursion of Ae. albopictus and pyrethroid resistance have so far been prevented, almost nothing is known about Aedes populations in neighbouring Papua New Guinea (PNG). With pyrethroid resistance documented in parts of Indonesia but not in Australia, it is important to determine the distribution of susceptible and resistant Aedes populations in this region. Methods: The present study was aimed at assessing Aedes populations for insecticide resistance in Madang and Port Moresby, located on the north and south coasts of PNG, respectively. Mosquitoes were collected using ovitraps and reared in an insectary. Standard WHO bioassays using insecticide-treated filter papers were conducted on a total of 253 Ae. aegypti and 768 Ae. albopictus adult mosquitoes. Subsets of samples from both species (55 Ae. aegypti and 48 Ae. albopictus) were screened for knockdown resistance mutations in the voltage-sensitive sodium channel (Vssc) gene, the target site of pyrethroid insecticides. Results: High levels of resistance against pyrethroids were identified in Ae. aegypti from Madang and Port Moresby. Aedes albopictus exhibited susceptibility to pyrethroids, but moderate levels of resistance to DDT. Mutations associated with pyrethroid resistance were detected in all Ae. aegypti samples screened. Some genotypes found in the present study had been observed previously in Indonesia. No Vssc mutations associated with pyrethroid resistance were found in the Ae. albopictus samples. Conclusions: To our knowledge, this is the first report of pyrethroid resistance in Ae. aegypti mosquitoes in PNG. Interestingly, usage of insecticides in PNG is low, apart from long-lasting insecticidal nets distributed for malaria control. Further investigations on how these resistant Ae. aegypti mosquito populations arose in PNG and how they are being sustained are warranted

Vector composition, abundance, biting patterns and malaria transmission intensity in Madang, Papua New Guinea: assessment after 7 years of an LLIN-based malaria control programme

Background: A malaria control programme based on distribution of long-lasting insecticidal bed nets (LLINs) and artemisinin combination therapy began in Papua New Guinea in 2009. After implementation of the programme, substantial reductions in vector abundance and malaria transmission intensity occurred. The research reported here investigated whether these reductions remained after seven years of sustained effort. Methods: All-night (18:00 to 06:00) mosquito collections were conducted using human landing catches and barrier screen methods in four villages of Madang Province between September 2016 and March 2017. Anopheles species identification and sporozoite infection with Plasmodium vivax and Plasmodium falciparum were determined with molecular methods. Vector composition was expressed as the relative proportion of different species in villages, and vector abundance was quantified as the number of mosquitoes per barrier screen-night and per person-night. Transmission intensity was quantified as the number of sporozoite-infective vector bites per person-night. Results: Five Anopheles species were present, but vector composition varied greatly among villages. Anopheles koliensis, a strongly anthropophilic species was the most prevalent in Bulal, Matukar and Wasab villages, constituting 63.7–73.8% of all Anopheles, but in Megiar Anopheles farauti was the most prevalent species (97.6%). Vector abundance varied among villages (ranging from 2.8 to 72.3 Anopheles per screen-night and 2.2–31.1 Anopheles per person-night), and spatially within villages. Malaria transmission intensity varied among the villages, with values ranging from 0.03 to 0.5 infective Anopheles bites per person-night. Most (54.1–75.1%) of the Anopheles bites occurred outdoors, with a substantial proportion (25.5–50.8%) occurring before 22:00. Conclusion: The estimates of vector abundance and transmission intensity in the current study were comparable to or higher than estimates in the same villages in 2010–2012, indicating impeded programme effectiveness. Outdoor and early biting behaviours of vectors are some of the likely explanatory factors. Heterogeneity in vector composition, abundance and distribution among and within villages challenge malaria control programmes and must be considered when planning them

Asia-Pacific ICEMR: Understanding Malaria Transmission to Accelerate Malaria Elimination in the Asia Pacific Region

Gaining an in-depth understanding of malaria transmission requires integrated, multifaceted research approaches. The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) is applying specifically developed molecular and immunological assays, in-depth entomological assessments, and advanced statistical and mathematical modeling approaches to a rich series of longitudinal cohort and cross-sectional studies in Papua New Guinea and Cambodia. This is revealing both the essential contribution of forest-based transmission and the particular challenges posed by Plasmodium vivax to malaria elimination in Cambodia. In Papua New Guinea, these studies document the complex host–vector–parasite interactions that are underlying both the stunning reductions in malaria burden from 2006 to 2014 and the significant resurgence in transmission in 2016 to 2018. Here we describe the novel analytical, surveillance, molecular, and immunological tools that are being applied in our ongoing Asia-Pacific ICEMR research program

Plasticity of host selection by malaria vectors of Papua New Guinea

Background Host selection is an important determinant of vectorial capacity because malaria transmission increases when mosquitoes feed more on humans than non-humans. Host selection also affects the outcome of long-lasting insecticidal nets (LLIN). Despite the recent nationwide implementation of LLIN-based malaria control program in Papua New Guinea (PNG), little is known about the host selection of the local Anopheles vectors. This study investigated the host selection of Anopheles vectors in PNG. Methods Blood-engorged mosquitoes were sampled using the barrier screen method and blood meals analyzed for vertebrate host source with PCR-amplification of the mitochondrial cytochrome b gene. Abundance of common hosts was estimated in surveys. The test of homogeneity of proportions and the Manly resource selection ratio were used to determine if hosts were selected in proportion to their abundance. Results Two thousand four hundred and forty blood fed Anopheles females of seven species were sampled from five villages in Madang, PNG. Of 2,142 samples tested, 2,061 (96.2%) yielded a definitive host source; all were human, pig, or dog. Hosts were not selected in proportion to their abundance, but rather were under-selected or over-selected by the mosquitoes. Four species, Anopheles farauti (sensu stricto) (s.s.), Anopheles punctulatus (s.s.), Anopheles farauti no. 4 and Anopheles longirostris, over-selected humans in villages with low LLIN usage, but over-selected pigs in villages with high LLIN usage. Anopheles koliensis consistently over-selected humans despite high LLIN usage, and Anopheles bancroftii over-selected pigs. Conclusions The plasticity of host selection of an Anopheles species depends on its opportunistic, anthropophilic or zoophilic behavior, and on the extent of host availability and LLIN usage where the mosquitoes forage for hosts. The high anthropophily of An. koliensis increases the likelihood of contacting the LLIN inside houses. This allows its population size to be reduced to levels insufficient to support transmission. In contrast, by feeding on alternative hosts the likelihood of the opportunistic species to contact LLIN is lower, making them difficult to control. By maintaining high population size, the proportion that feed on humans outdoors can sustain residual transmission despite high LLIN usage in the village

Infectivity of Symptomatic Malaria Patients to Anopheles farauti Colony Mosquitoes in Papua New Guinea

Plasmodium transmission from humans to mosquitoes is an understudied bottleneck in the transmission of malaria. Direct membrane feeding assays (DMFA) allow detailed malaria transmission studies from humans to mosquitoes. Especially for Plasmodium vivax, which cannot be cultured long-term under laboratory conditions, implementation of DMFAs requires proximity to P. vivax endemic areas. In this study, we investigated the infectivity of symptomatic Plasmodium infections to Anopheles farauti colony mosquitoes in Papua New Guinea (PNG). A total of 182 DMFAs were performed with venous blood collected from rapid diagnostic test (RDT) positive symptomatic malaria patients and subsequently analysed by light microscopy and quantitative real time polymerase chain reaction (qPCR). DMFAs resulted in mosquito infections in 20.9% (38/182) of cases. By light microscopy and qPCR, 10 – 11% of P. falciparum and 32 – 44% of P. vivax positive individuals infected An. farauti. Fifty-eight percent of P. vivax and 15% of P. falciparum gametocytaemic infections infected An farauti

Genetic differentiation and bottleneck effects in the malaria vectors Anopheles farauti and Anopheles punctulatus after an LLIN‐based vector control program in Papua New Guinea

Implementation of long‐lasting insecticide‐treated net (LLIN) programs to control human malaria transmission leads to substantial reductions in the abundance of Anopheles mosquitoes, but the impact on the population genetic structure of the malaria vectors is poorly known, nor has it been investigated in Papua New Guinea, where malaria is highly endemic and where several species of Anopheles have vector roles. Here, we applied Wright's F‐statistic, analysis of molecular variance, Bayesian structure analysis, and discriminant analysis of principle components to microsatellite genotype data to analyze the population genetic structure of Anopheles farauti between and within the northern and southern lowland plains and of Anopheles punctulatus within the northern plain of Papua New Guinea after such a program. Bottleneck effects in the two malaria vectors were analyzed using Luikart and Cornuet's tests of heterozygosity. A large, panmictic population of An. punctulatus pre‐LLIN program diverged into two subregional populations corresponding to Madang and East Sepik provinces post‐LLIN distribution and experienced a genetic bottleneck during this process. By contrast, the An. farauti population existed as two regional populations isolated by mountain ranges pre‐LLIN, a genetic structure that persisted after the distribution of LLINs with no further geographic differentiation nor evidence of a genetic bottleneck. These findings show the differential response of populations of different vector species to interventions, which has implications for program sustainability and gene flow

Decreased bioefficacy of long-lasting insecticidal nets and the resurgence of malaria in Papua New Guinea

Papua New Guinea (PNG) has the highest malaria transmission outside of Africa. Longlasting insecticidal nets (LLINs) are believed to have helped to reduce average malaria prevalence in PNG from 16% in 2008 to 1% in 2014. Since 2015 malaria in PNG has resurged significantly. Here, we present observations documenting decreased bioefficacy of unused LLINs with manufacturing dates between 2013 and 2019 collected from villages and LLIN distributors in PNG. Specifically, we show that of n = 167 tested LLINs manufactured after 2013, only 17% are fulfilling the required World Health Organisation bioefficacy standards of ≥ 80% 24 h mortality or ≥ 95% 60 min knockdown in bioassays with pyrethroid susceptible Anopheles farauti mosquitoes. In contrast, all (100%, n = 25) LLINs with manufacturing dates prior to 2013 are meeting these bioefficacy standards. These results suggest that decreased bioefficacy of LLINs is contributing to the malaria resurgence in PNG and of LLIN quality is warranted

Disruption of Mouse Cenpj, a Regulator of Centriole Biogenesis, Phenocopies Seckel Syndrome

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpjtm/tm) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpjtm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpjtm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome