De novo transcriptome sequencing and annotation of the Antarctic polychaete Microspio moorei (Spionidae) with its characterization of the heat stress-related proteins (HSP, SOD & CAT).

We present a de novo transcriptome assembly for the non-model Antarctic polychaete worm Microspio moorei (Spionidae) collected during Antarctic field expedition in Fildes Bay, King George Island, Antarctic Peninsula, in 2017. Here, we report the first transcriptome reference array for Microspio spp. The gene sequences of the spionid worm were annotated from a wide range of functions (i.e., biological, and metabolic processes, catalytic processes, and catalytic activity). HSP70, HSP90 SOD and CAT families were compared to reported annelid transcriptomes and proteomes. The phylogenetic analysis using COI, 16S, and 18S markers effectively clusters the species within the family. However, it also casts uncertainty on the monophyletic nature of the Microspio genera, indicating the necessity for additional data and potentially requiring a reevaluation of its grouping. Within these protein families, 3D model software was used to create one representative of their protein structures. Structural predictions were compared with related reported annelids living at different temperatures and a human X-ray reference. We found structural differences (RMSE >1.8) between the human HSP proteins but no significant differences between the polychaete-predicted proteins (RMSE <1.2). These results encourage further research of heat stress-related proteins, the development of genetic markers for climate change-induced temperature stress, and the study of the underlying mechanisms of the heat response. Moreover, these results motivate the extension of these findings to congeneric species.

Two new species of spionids from the genera Apoprionospio Foster, 1969 and Prionospio Malmgren, 1867 (Annelida: Spionidae) from the Colombian Caribbean.

Two species of genera Apoprionospio and Prionospio are described from the Colombian Caribbean. Apoprionospio sanguarensis sp. nov. and Prionospio gabriellae sp. nov. are new to science. Apoprionospio sanguarensis sp. nov. is distinguished from the three previously described Apoprionospio species by having the smallest body size, triangular prostomium, three pairs of eyes, the branchial pairs 1-3 are cirriform, wrinkled, the anterior notochaetae arranged from chaetiger 2 onwards arranged in three rows, and all hooded hooks lack secondary hoods. Prionospio gabriellae sp. nov. is distinguished from all Prionospio species with five branchial pairs by having dorsal crests across the dorsum on chaetigers 16-25. Closely related species are P. heterobranchia Moore, 1907 and P. caribensis Delgado-Blas, 2014 and Prionospio gabriellae sp. nov. differs from both species in that it has a small medial peak in the dorsal region of prostomium, the first neuropodial lamellae is very small, the shaped of branchial pairs 2 and 3 are cirriform, neuropodial postchaetal lamellae on chaetiger 2 is square, all chaetae of chaetiger 1 are arranged in a row. We propose raising the status of Apoprionospio dayi japonica Imajima, 1989 to the species level as A. japonica Imajima, 1989. A key and a table detailing the most relevant characters of Apoprionospio species and a key to all species of Prionospio with five pairs of branchiae are provided.

Geometric morphometrics of nine field isolates of Aedes aegypti with different resistance levels to lambda-cyhalothrin and relative fitness of one artificially selected for resistance.

Aedes aegypti, a mosquito closely associated with humans, is the principal vector of dengue virus which currently infects about 400 million people worldwide. Because there is no way to prevent infection, public health policies focus on vector control; but insecticide-resistance threatens them. However, most insecticide-resistant mosquito populations exhibit fitness costs in absence of insecticides, although these costs vary. Research on components of fitness that vary with insecticide-resistance can help to develop policies for effective integrated management and control. We investigated the relationships in wing size, wing shape, and natural resistance levels to lambda-cyhalothrin of nine field isolates. Also we chose one of these isolates to select in lab for resistance to the insecticide. The main life-traits parameters were assessed to investigate the possible fitness cost and its association with wing size and shape. We found that wing shape, more than wing size, was strongly correlated with resistance levels to lambda-cyhalothrin in field isolates, but founder effects of culture in the laboratory seem to change wing shape (and also wing size) more easily than artificial selection for resistance to that insecticide. Moreover, significant fitness costs were observed in response to insecticide-resistance as proved by the diminished fecundity and survival of females in the selected line and the reversion to susceptibility in 20 generations of the non-selected line. As a practical consequence, we think, mosquito control programs could benefit from this knowledge in implementing efficient strategies to prevent the evolution of resistance. In particular, the knowledge of reversion to susceptibility is important because it can help in planning better strategies of insecticide use to keep useful the few insecticide-molecules currently available.

Temephos resistance in Aedes aegypti in Colombia compromises dengue vector control.

BACKGROUND: Control and prevention of dengue relies heavily on the application of insecticides to control dengue vector mosquitoes. In Colombia, application of the larvicide temephos to the aquatic breeding sites of Aedes aegypti is a key part of the dengue control strategy. Resistance to temephos was recently detected in the dengue-endemic city of Cucuta, leading to questions about its efficacy as a control tool. Here, we characterize the underlying mechanisms and estimate the operational impact of this resistance. METHODOLOGY/PRINCIPAL FINDINGS: Larval bioassays of Ae. aegypti larvae from Cucuta determined the temephos LC50 to be 0.066 ppm (95% CI 0.06-0.074), approximately 15× higher than the value obtained from a susceptible laboratory colony. The efficacy of the field dose of temephos at killing this resistant Cucuta population was greatly reduced, with mortality rates <80% two weeks after application and <50% after 4 weeks. Neither biochemical assays nor partial sequencing of the ace-1 gene implicated target site resistance as the primary resistance mechanism. Synergism assays and microarray analysis suggested that metabolic mechanisms were most likely responsible for the temephos resistance. Interestingly, although the greatest synergism was observed with the carboxylesterase inhibitor, DEF, the primary candidate genes from the microarray analysis, and confirmed by quantitative PCR, were cytochrome P450 oxidases, notably CYP6N12, CYP6F3 and CYP6M11. CONCLUSIONS/SIGNIFICANCE: In Colombia, resistance to temephos in Ae. aegypti compromises the duration of its effect as a vector control tool. Several candidate genes potentially responsible for metabolic resistance to temephos were identified. Given the limited number of insecticides that are approved for vector control, future chemical-based control strategies should take into account the mechanisms underlying the resistance to discern which insecticides would likely lead to the greatest control efficacy while minimizing further selection of resistant phenotypes.

Insecticide resistance status of Aedes aegypti (L.) from Colombia.

BACKGROUND: To evaluate the insecticide susceptibility status of Aedes aegypti (L.) in Colombia, and as part of the National Network of Insecticide Resistance Surveillance, 12 mosquito populations were assessed for resistance to pyrethroids, organophosphates and DDT. Bioassays were performed using WHO and CDC methodologies. The underlying resistance mechanisms were investigated through biochemical assays and RT-PCR. RESULTS: All mosquito populations were susceptible to malathion, deltamethrin and cyfluthrin, and highly resistant to DDT and etofenprox. Resistance to lambda-cyhalothrin, permethrin and fenitrothion ranged from moderate to high in some populations from Chocó and Putumayo states. In Antioquia state, the Santa Fe population was resistant to fenitrothion. Biochemical assays showed high levels of both cytochrome P450 monooxygenases (CYP) and non-specific esterases (NSE) in some of the fenitrothion- and pyrethroid-resistant populations. All populations showed high levels of glutathione-S-transferase (GST) activity. GSTe2 gene was found overexpressed in DDT-resistant populations compared with Rockefeller susceptible strain. CONCLUSIONS: Differences in insecticide resistance status were observed between insecticides and localities. Although the biochemical assay results suggest that CYP and NSE could play an important role in the pyrethroid and fenitrothion resistance detected, other mechanisms remain to be investigated, including knockdown resistance. Resistance to DDT was high in all populations, and GST activity is probably the main enzymatic mechanism associated with this resistance. The results of this study provide baseline data on insecticide resistance in Colombian A. aegypti populations, and will allow comparison of changes in susceptibility status in this vector over time.

Pyrethroid and organophosphates resistance in Anopheles (N.) nuneztovari Gabaldón populations from malaria endemic areas in Colombia.

Field populations of Colombian malaria vector Anopheles (N.) nuneztovari were studied using World Health Organization (WHO) and Center for Disease Control and Prevention (CDC) bioassay techniques and through the use of biochemical microplate-based assays for resistance enzymes. Insecticides evaluated included the pyrethroids lambda-cyhalothrin and deltamethrin, organophosphates malathion and fenitrothion, and the organochlorine dichlorodiphenyltrichloroethane (DDT). Study sites selected were based upon malaria incidence, vector presence, and control activities in Colombia. Early stage selection for reduced susceptibility was observed in the bioassays for some locations. Data from the WHO and CDC bioassay methods were broadly consistent, with some differences noted. Evidence is presented for low-level initial selection of some resistance mechanisms such as mixed-function oxidases and modified acetylcholinesterase. Data from the site Encharcazón implies that selection for DDT-pyrethroid cross-resistance has occurred, though not likely at a level that currently threatens vector control by either class of insecticides, and further implies that knockdown resistance (kdr) may be present in those populations. Further studies using synergists and development of a kdr-specific assay for A. nuneztovari thus become priorities. The resistance levels to lambda-cyhalothrin and deltamethrin found in the Encharcazón population are of concern since these two insecticides are currently used for both indoor spraying and treated nets. In addition, the resistance to fenitrothion, the indoor spray insecticide mostly used for this species due to their exophilic behavior, found in the El Zulia population, makes urgent to find alternatives for chemical control in these areas. These data provide the initial baselines for insecticide susceptibility profiles for A. nuneztovari in Colombia and the first report of insecticide resistance in this vector.

Mixed-function oxidases and esterases associated with cross-resistance between DDT and lambda-cyhalothrin in Anopheles darlingi Root 1926 populations from Colombia.

In order to establish the insecticide susceptibility status for Anopheles darlingi in Colombia, and as part of the National Network on Insecticide Resistance Surveillance, five populations of insects from three Colombian states were evaluated. Standardised WHO and CDC bottle bioassays, in addition to microplate biochemical assays, were conducted. Populations with mortality rates below 80% in the bioassays were considered resistant. All field populations were susceptible to deltamethrin, permethrin, malathion and fenitrothion. Resistance to lambda-cyhalothrin and DDT was detected in the Amé-Beté population using both bioassay methods with mortality rates of 65-75%. Enzyme levels related to insecticide resistance, including mixed function oxidases (MFO), non-specific esterases (NSE), glutathione S-transferases and modified acetylcholinesterase were evaluated in all populations and compared with a susceptible natural strain. Only mosquitoes from Amé-Beté presented significantly increased levels of both MFO and NSE, consistent with the low mortalities found in this population. The continued use of lambda-cyhalothrin for An. darlingi control in this locality has resulted in a natural resistance to this insecticide. In addition, DDT resistance is still present in this population, although this insecticide has not been used in Colombia since 1992. Increased metabolism through MFO and NSE may be involved in cross-resistance between lambda-cyhalothrin and DDT, although kdr-type nerve insensitivity cannot be discarded as a possible hypothesis. Additional research, including development of a kdr specific assay for An. darlingi should be conducted in future studies. Our data demonstrates the urgent need to develop local insecticide resistance management and surveillance programs throughout Colombia.

Mixed-function oxidases and esterases associated with cross-resistance between DDT and lambda-cyhalothrin in Anopheles darlingi Root 1926 populations from Colombia

In order to establish the insecticide susceptibility status for Anopheles darlingi in Colombia, and as part of the National Network on Insecticide Resistance Surveillance, five populations of insects from three Colombian states were evaluated. Standardised WHO and CDC bottle bioassays, in addition to microplate biochemical assays, were conducted. Populations with mortality rates below 80 percent in the bioassays were considered resistant. All field populations were susceptible to deltamethrin, permethrin, malathion and fenitrothion. Resistance to lambda-cyhalothrin and DDT was detected in the Amé-Beté population using both bioassay methods with mortality rates of 65-75 percent. Enzyme levels related to insecticide resistance, including mixed function oxidases (MFO), non-specific esterases (NSE), glutathione S-transferases and modified acetylcholinesterase were evaluated in all populations and compared with a susceptible natural strain. Only mosquitoes from Amé-Beté presented significantly increased levels of both MFO and NSE, consistent with the low mortalities found in this population. The continued use of lambda-cyhalothrin for An. darlingi control in this locality has resulted in a natural resistance to this insecticide. In addition, DDT resistance is still present in this population, although this insecticide has not been used in Colombia since 1992. Increased metabolism through MFO and NSE may be involved in cross-resistance between lambda-cyhalothrin and DDT, although kdr-type nerve insensitivity cannot be discarded as a possible hypothesis. Additional research, including development of a kdr specific assay for An. darlingi should be conducted in future studies. Our data demonstrates the urgent need to develop local insecticide resistance management and surveillance programs throughout Colombia.