The copepod Diaptomus sanguineus begins diapause in permanent ponds in late March as an adaptation to avoid summer fish predation. During a study of copepod populations in two Rhode Island ponds, a severe drought dried one pond killing all fish. The second (control) pond did not dry, and no fish were killed. Before the drought copepods in the two ponds entered diapause on nearly the same date. After the drought, the timing of diapause shifted to later in the year in the pond that had lost its fish, while no shift occurred in the control pond. The direction of this shift in the onset of diapause is that expected had the copepods been released from natural selection for early spring diapause imposed by summer fish predation.There are surprisingly few examples of adaptive evolution by natural selection in wild populations. Indeed, the best documented examples of selection in the field are of population responses to human disturbance (1-3). Natural disturbances or catastrophes that might lead to evolutionary change are difficult or impossible to predict and so the locations of appropriate experimental and control areas cannot be anticipated nor can studies of initial conditions be planned.Here we report observations involving two populations of the planktonic freshwater copepod Diaptomus sanguineus that were fortuitously under investigation when a natural disaster removed an important agent of selection from one population while leaving it unaltered in the other. The result was a rapid directional shift in the life history of the affected population while the control population remained unchanged. STUDY SITES AND METHODSDiaptomus sanguineus makes two kinds of eggs: (i) subitaneous eggs that hatch rapidly and (ii) diapausing eggs that rest in the sediments for an extended period before hatching (4, 5). Egg clutches are of one or the other type. Mixed clutches are never produced (5). The timing of the seasonal shift between egg types has been followed since 1979 for D. sanguineus populations inhabiting two small Rhode Island ponds. Bullhead Pond (2.3 ha, surface area; 4 m, maximum depth) and Little Bullhead Pond (0.1 ha; 2.4 m) lie in separate drainages within 200 m of each other on private land. In 1979 egg type was determined as described (4, 5) on groups of 20 ovigerous females collected biweekly. In subsequent years determinations were made on groups of 24 ovigerous females collected weekly, except in 1980 when no determinations were made.To ascertain the extent to which photoperiod controls D. sanguineus reproductive phenology, we collected first stage nauplii (newly hatched) in the field and reared them to sexual maturity (about 30 days) in the laboratory in four controlled environment chambers at 9 + 1°C. Photoperiods were chosen (Table 1) to bracket the day length at the time of year that most copepods begin producing diapausing eggs.

BackgroundThe native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes.ResultsBeta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2 % of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82 % of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes.ConclusionsBacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0475-8) contains supplementary material, which is available to authorized users.

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.

An urgent need exists for new agents to control mosquito vectors of disease. Mosquito larvicides based on the bacteria Bacillus thuringiensis subsp. israelensis (Bti) or B. sphaericus (Bs) are effective in many habitats, but use is limited by their high cost. Moreover, mosquito resistance evolves rapidly to Bs where it is used intensively. The efficacy of these bacteria is due to a binary protein (BsB) in Bs and four proteins (Cry4A, Cry4B, Cry11A, and Cyt1A) in Bti. Here we report the use of cyt1A promoters and a 5' mRNA stabilizing sequence to synthesize high levels of Bs2362 binary toxin in Bti strains. The recombinant BtiIPS-82/BsB showed high potency against fourth instars of Culex quinquefasciatus, a vector of West Nile virus, being 21-fold as potent as BtiIPS-82, and 32-fold as potent as Bs2362. Similar improved efficacy was obtained against larvae of Cx. tarsalis. Moreover, BtiIPS-82/BsB suppressed resistance to Bs2362 in Cx. quinquefasciatus.

We studied skewed adult copepod sex ratios in two ponds in an attempt to understand the ecological causes and evolutionary consequences of this life-history phenomenon.In ponds containing populations of zooplanktivorous sunfish, female copepods suffered higher mortality rates than males, while in ponds lacking these predators, male and female mortality rates were similar. Field and laboratory experiments showed that females, especially those carrying eggs, were more visible than males and that added visibility accounted for higher predation by fish on these individuals.We calculated the cost of carrying eggs in terms of the decreased population growth rate it produces, and then estimated the magnitude of egg mortality necessary to select for the egg-carrying strategy. Eggs not carried would have to suffer at .least 58% mortality for egg carrying to evolve in the presence of selective fish predation. The response of the copepods to these strong selective pressures lies in a third direction, the production of diapausing eggs.