Invading new climates at what cost? Ontogenetic differences in the thermal dependence of metabolic rate in an invasive amphibian.

Global warming can either promote or constrain the invasive potential of alien species. In ectotherm invaders that exhibit a complex life cycle, success is inherently dependent on the capacity of each developmental stage to cope with environmental change. This is particularly relevant for invasive anurans, which disperse on land while requiring water for reproduction. However, it remains unknown how the different life stages respond in terms of energy expenditure under different climate change scenarios. We here quantified the oxygen uptake of frogs at rest (a proxy of the standard metabolic rate) in the aquatic phase (at the tadpole and climax, i.e. during metamorphosis, stages) and in the terrestrial phase (metamorphosed stage) at three environmental temperatures. To do so, we used marsh frogs (Pelophylax ridibundus), an amphibian with the largest invasive range within the palearctic realm and for which their adaptation to global warming might be key to their invasion success. Beyond an increase of metabolic rate with temperature, our data show variation in thermal adaptation across life stages and a higher metabolic cost during metamorphosis. These results suggest that the cost to shift habitat and face changes in temperature may be a constraint on the invasive potential of species with a complex life cycle which may be particularly vulnerable during metamorphosis.

Assessment of toxicity, genotoxicity and oxidative stress in Fejervarya limnocharis exposed to tributyltin.

Tributyltin (TBT) is widely used in various commercial applications due to its biocidal properties. Toxicological and genotoxicological data on TBT exposure to amphibians is insufficient. Our study aimed to determine the acute toxicity and genotoxic potential of TBT in Fejervarya limnocharis tadpoles. Furthermore, oxidative stress was also investigated in TBT-treated tadpoles. Tadpoles of Gosner stage (26-30) were screened and subjected to increasing concentrations of TBT (0, 3, 7, 11, 15, 19, 23 µg/L) for determining the LC50 values for 24 h, 48 h, 72 h, and 96 h. LC50 values of TBT for 24 h, 48 h, 72 h, and 96 h were found to be 19.45, 15.07, 13.12, and 11.84 µg/L respectively. Based on the 96 h LC50 value (11.84 µg/L), tadpoles were exposed to different sub-lethal concentrations of TBT for the evaluation of its genotoxic potential and effects on oxidative balance. The role of TBT on survivability, growth, and time to metamorphosis was also assessed. TBT exposure significantly altered the life history traits measured, increased mortality, and delayed the time taken to metamorphosis. Results indicated significant induction of micronucleus (MN, p < 0.001) and other erythrocytic nuclear aberrations (ENA, p < 0.01) in the TBT-treated groups. Significant alterations in comet parameters and oxidative balance were also observed in the treated groups. The present study findings might add to the cause of the gradual population decline seen in the amphibians. This study also demonstrates the alteration of the life-history traits, oxidative balance, and DNA damage upon TBT exposure which can have long-term consequences for the anuran amphibian F. limnocharis.

Effect assessment of reclaimed waters and carbamazepine exposure on the thyroid axis of Xenopus laevis: Gene expression modifications.

Reclaimed water (RW) obtained from wastewater treatment plants (WWTP) is used for irrigation, groundwater recharge, among other potential uses. Although most pollutants are removed, traces of them are frequently found, which can affect organisms and alter the environment. The presence of a myriad of contaminants in RW makes it a complex mixture with very diverse effects and interactions. A previous study, in which tadpoles were exposed to RW and RW spiked with Carbamazepine (CBZ), presented slight thyroid gland stimulation, as suggested by the development acceleration of tadpoles and histological findings in the gland provoked by RW, regardless of the CBZ concentration. To complement this study, the present work analysed the putative molecular working mechanism by selecting six genes coding for the thyroid-stimulating hormone (TSHß), thyroid hormone metabolising enzymes (DIO2, DIO3), thyroid receptors (THRA, THRB), and a thyroid hormone-induced DNA binding protein (Kfl9). Transcriptional activity was studied by Real-Time PCR (RT-PCR) in brains, hind limbs, and tails on exposure days 1, 7, and 21. No significant differences were observed between treatments for each time point, but slight alterations were noted when the time response was analysed. The obtained results indicate that the effects of RW or RW spiked with CBZ are negligible for the genes analysed during the selected exposure periods.

Mechanism of threshold size assessment: Metamorphosis is triggered by the TGF-beta/Activin ligand Myoglianin.

Although the mechanisms that control growth are now well understood, the mechanism by which animals assess their body size remains one of the great puzzles in biology. The final larval instar of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a threshold size. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to change depending on the amount of exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower threshold size. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.

Effect assessment of reclaimed water and carbamazepine exposure on the thyroid axis of X. laevis: Apical and histological effects.

There is increasing environmental concern about the constant presence of pharmaceuticals and personal care products (PPCPs) in surface water, generally attributed to water discharge from wastewater treatment plants (WWTPs) that are unable to completely remove these compounds. The slight, but continuous, presence of these contaminants in reclaimed water (RW) poses a risk of chronic and sublethal toxicity, and the thyroid axis can likely be a target of many of these PPCPs. In this work, we addressed the effects of RW on the Xenopus laevis thyroid system. The Amphibian Metamorphosis Assay (AMA test) was used with modifications by exposing X. laevis tadpoles to RW samples, and to RW spiked with carbamazepine (CBZ) at 100 and 1000 higher than the average levels environmentally relevant (RW 100× and RW 1000×, respectively). Carbamazepine was selected because it is considered a marker of anthropogenic pollution and could have a potential effect on the thyroid axis. The morphological endpoints and histological alterations to the thyroid gland were evaluated. The results suggested the stimulation of the thyroid gland from exposures to the RW samples, supported by tadpoles' accelerated development and by the histological alterations observed in the thyroid gland. Developmental acceleration was also seen in the tadpoles exposed to the RW-100× and -1000× samples at comparable levels to those seen in exposures to RW samples alone. Hence CBZ did not seem to increase the effects of RW on the thyroid axis. Overall, our results suggested endocrine effects of these RW samples regardless of the CBZ concentration.

Assessment of antioxidant system, cholinesterase activity and histopathology in Lithobates catesbeianus tadpoles exposed to water from an urban stream.

Anthropogenic activities promote changes in community structure and decrease the species abundance of amphibians. The aim of this study was to assess potential alterations in the antioxidant system and cholinesterase activity, histopathological and oxidative damage in Lithobates catesbeianus tadpoles exposed to water from the Cascavel River, in Southern Brazil. Water samples (140 L each) were collected from the headwater, urban and rural areas of the river. Tadpoles were acclimated for seven days. After acclimatization tadpoles were reared in water from the river, except for the control aquarium. After seven days, a portion of the liver was removed and prepared for cholinesterase (ChE), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) analysis; another part of the tissue was prepared for histological examination. An elevation of CAT activity was observed for water from both urban and rural environments. A decrease in LPO reaction was detected, mainly among the tadpoles exposed to water from the rural area. These alternations can cause delay the metamorphosis and lead to metabolic dysfunction, interfering in survival capacity and diminishing, not only individual fitness, but that of the whole population.

Metabolic costs of altered growth trajectories across life transitions in amphibians.

Climate change is causing increases in temperature and in the frequency of extreme weather events. Under this scenario, organisms should maintain or develop strategies to cope with environmental fluctuations, such as the capacity to modify growth trajectories. However, altering growth can have negative consequences for organisms' fitness. Here, we investigated the metabolic alterations induced by compensatory growth during the larval development of the common frog (Rana temporaria), quantifying changes in oxidative stress, corticosterone levels and telomere length. We induced compensatory growth responses by exposing frog embryos to cold conditions (i.e. a 'false spring' scenario), which cause a delay in hatching. Once hatched, we reared larvae at two different photoperiods (24:0, representing the natural photoperiod of larvae, and 18:6) to test also for the interactive effects of light on growth responses. Larvae experiencing delayed hatching showed fast compensatory responses and reached larger size at metamorphosis. Larvae shortened their developmental period in response to delayed hatching. Non-permanent light conditions resulted in relaxed growth compared with larvae reared under permanent light conditions, which grew at their natural photoperiod and closer to their maximal rates. Growth responses altered the redox status and corticosterone levels of larvae. These physiological changes were developmental stage-dependent and mainly affected by photoperiod conditions. At catch-up, larvae reared at 18:6 light:dark cycles showed higher antioxidant activities and glucocorticoid secretion. On the contrary, larvae reared at 24:0 developed at higher rates without altering their oxidative status, likely an adaptation to grow under very restricting seasonal conditions at early life. At metamorphosis, compensatory responses induced higher cellular antioxidant activities probably caused by enhanced metabolism. Telomere length remained unaltered by experimental treatments but apparently tended to elongate across larval ontogeny, which would be a first evidence of telomere lengthening across metamorphosis. Under the forecasted increase in extreme climatic events, adjusting growth and developmental rates to the dynamics of environmental fluctuations may be essential for survival, but it can carry metabolic costs and affect later performance. Understanding the implications of such costs will be essential to properly estimate the impact of climate change on wild animals.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life-history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967-1977. © 2019 SETAC.

Evaluating the role of fish as surrogates for amphibians in pesticide ecological risk assessment.

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (rs) = 0.88], body weight (rs = 0.86), and length (rs = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages.

Is normalized hindlimb length measurement in assessment of thyroid disruption in the amphibian metamorphosis assay relevant?

The amphibian metamorphosis assay represents an OECD Level 3 and EDSP Tier 1 ecotoxicity test assessing thyroid activity of chemicals in African clawed frog (Xenopus laevis). To evaluate the effectiveness of snout-vent length (SVL) normalization of hindlimb length (HLL), correlation between the HLL and SVL or body weight was evaluated in the control groups of 10 individual studies from three laboratories. Two studies required separate analysis of the Nieuwkoop-Faber (NF) stage ≤60 and >60 animals creating a total of 12 data sets. On study day 7, significant positive correlation between HLL and SVL or body weight was observed in eight and seven of the 10 data sets, respectively (r = 0.608-0.843 and 0.583-0.876). On study day 21, significant positive correlation between HLL and SVL or body weight was found in three and four of the 12 data sets, respectively (r = 0.452, 0.480 and 0.553 and r = 0.621, 0.546, 0.564 and 0.378). Significant positive correlation between HLL and SVL was found in three of five studies, including ≤NF stage 60 data (r = 0.564, 0.546 and 0.621). In one of eight studies, including >NF stage 60 data, the positive correlation between HLL and body weight was determined (r = 0.378). Negative or no correlation between HLL and SVL or body weight was found in the other late stage data sets. Therefore, use of SVL-normalized HLL to assess thyroid-mediated effects in X. laevis tadpoles is not warranted. HL stage relative to body stage should be considered.

Effects of nonylphenols on embryonic development and metamorphosis of Xenopus laevis: FETAX and amphibian metamorphosis toxicity test (OECD TG231).

Nonylphenols (NPs) are a group of endocrine-disrupting surfactants that mimic estrogen. To determine the developmental toxicity and thyroid-disrupting effect of NPs, the effects of exposure to nonylphenol (NP), 4-nonylphenol (4-NP), and nonylphenol ethoxylate (NP-12) were examined according to the frog embryo teratogenesis assay-Xenopus (FETAX) and Organization for Economic Co-operation and Development test guidelines 231 (TG231). In FETAX, the LC50 values of NP, 4-NP, and NP-12 were 59.14 mg/L, 10.13 mg/L, and 14.60 mg/L, respectively. At 10.0 mg/L, NP, 4-NP, and NP-12 significantly decreased the total length of tadpoles, and NP and 4-NP increased gut malformation and bent tails. In surviving tadpoles, the EC50 values for malformation of NP, 4-NP, and NP-12 were 4.66, 6.51, and 13.08 mg/L, respectively. The teratogenic indices of NP, 4-NP, and NP-12 were 12.69, 1.56, and 1.08, respectively, suggesting the teratogenic potential of NP and 4-NP. In a range-finder assay for TG231, the 96-h LC50 values of NP, 4-NP, and NP-12 were 2.0, 2.0, and 10.57 mg/L, respectively. When NF stage 51 larvae were exposed for 21 days, larval growth was inhibited by NP, 4-NP, and NP-12 at 0.67, 0.07, and 0.37 mg/L, respectively. 4-NP at 0.07 mg/L accelerated the developmental stage and significantly increased hind limb length, while 0.67 mg/L 4-NP delayed the developmental stage and decreased hind limb length, suggesting a bimodal effect of 4-NP on metamorphosis. NP and NP-12 at test concentrations did not alter the larval stage, but NP-12 at 0.37 mg/L significantly decreased total length and tail length, suggesting growth inhibition in larvae. The total colloid area of thyroid follicles was significantly increased by 0.07 mg/L 4-NP but not by NP and NP-12, suggesting that 4-NP may interfere with thyroid function. Together, the developmental toxicity of NPs was in the following order: 4-NP, NP-12, and NP. 4-NP may alter metamorphosis driven by thyroid hormones in X. laevis.

Exogenous stress hormones alter energetic and nutrient costs of development and metamorphosis.

Variation in environmental conditions during larval life stages can shape development during critical windows and have lasting effects on the adult organism. Changes in larval developmental rates in response to environmental conditions, for example, can trade off with growth to determine body size and condition at metamorphosis, which can affect adult survival and fecundity. However, it is unclear how use of energy and nutrients shape trade-offs across life-stage transitions because no studies have quantified these costs of larval development and metamorphosis. We used an experimental approach to manipulate physiological stress in larval amphibians, along with respirometry and 13C-breath testing to quantify the energetic and nutritional costs of development and metamorphosis. Central to larval developmental responses to environmental conditions is the hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis, which regulates development, as well as energy homeostasis and stress responses across many taxa. Given these pleiotropic effects of HPA/I activity, manipulation of the HPA/I axis may provide insight into costs of metamorphosis. We measured the energetic and nutritional costs across the entire larval period and metamorphosis in a larval amphibian exposed to exogenous glucocorticoid (GC) hormones - the primary hormone secreted by the HPA/I axis. We measured metabolic rates and dry mass across larval ontogeny, and quantified lipid stores and nutrient oxidation via 13C-breath testing during metamorphosis, under control and GC-exposed conditions. Changes in dry mass match metamorphic states previously reported in the literature, but dynamics of metabolism were influenced by the transition from aquatic to terrestrial respiration. GC-treated larvae had lower dry mass, decreased fat stores and higher oxygen consumption during stages where controls were conserving energy. GC-treated larvae also oxidized greater amounts of 13C-labelled protein stores. These results provide evidence for a proximate cause of the physiological trade-off between larval growth and development, and provide insight into the energetic and nutrient costs that shape fitness trade-offs across life stages.

Variation in malathion sensitivity among populations of Blanchard's cricket frogs (Acris blanchardi) and implications for risk assessment.

Intraspecific variability in contaminant sensitivity could undermine risk assessments for nontarget organisms such as amphibians. To test how amphibian populations vary in tolerance to anticipated lethal and sublethal exposures to a pesticide, we exposed Blanchard's cricket frogs (Acris blanchardi) from 3 populations across a broad portion of their range to the insecticide malathion. Exposure in mesocosms to a nominal concentration of 1 mg/L (measured concentrations at 1 h and 24 h postaddition of 0.160 mg/L and 0.062 mg/L, respectively), a realistic direct-overspray scenario, reduced survival to metamorphosis by 43% relative to controls and revealed variation in tolerance among populations. Survival ranged from 74% for the most tolerant population to 18% for the least tolerant population, a 4.1-fold difference. Mass at metamorphosis and time to metamorphosis were unaffected. Although malathion reduced zooplankton abundance, it did not alter food resources (periphyton or phytoplankton relative abundance), or a suite of water-quality variables (pH, temperature, and dissolved oxygen). A 96-h time-to-death assay designed to isolate direct, lethal effects also revealed variation in tolerance among populations. Time to death (mean ± standard error) ranged from 2.4 ± 0.18 h for the least tolerant population to 17.8 ± 4.72 h for the most tolerant population, a 7.4-fold difference. However, relative sensitivities of populations differed in the mesocosm and laboratory studies, which differed in exposure concentrations, suggesting that populations tolerant of high concentrations can be more sensitive to lower concentrations. We suggest that direct overspray could reduce larval survival in the field for this species. Studies assessing the role of contaminants in declines or extrapolating to untested populations, especially across large geographical regions, should quantify the range of intraspecific variation. Risk assessors could address intraspecific variability directly by using an intraspecific uncertainty factor. Environ Toxicol Chem 2017;36:1917-1923. © 2016 SETAC.

Costs and Benefits of Underground Pupal Chambers Constructed by Insects: A Test Using Manduca sexta.

Many holometabolous insects metamorphose in belowground pupal chambers. Although the chambers may be elaborate and their construction costly, their functions are unknown. Using laboratory and field experiments, we examined the costs and functions of chambers made by the hawk moth Manduca sexta (Sphingidae). Costs were large in some circumstances; prepupal larvae lost up to 60% of their body mass when constructing chambers in dry soils. We tested three alternative hypotheses about what, if anything, chambers do for the individuals that make them: (1) chambers provide critical open space underground, allowing room for ecdysis and preventing soil from deforming the metamorphosing individual; (2) chambers raise the local relative humidity, so that cuticular and respiratory water losses are minimized; and (3) chamber walls prevent predators and pathogens from attacking. The data support the first hypothesis (about open space) and largely exclude the other two. These results provide a simple and potentially broad explanation for the evolution of chamber building in metamorphosing insects.

Assessment of toxicity test endpoints for freshwater mussel larvae (glochidia).

The objectives of the present study were to determine if the viability of freshwater mussel larvae (glochidia) is an ecologically relevant endpoint for toxicity tests and to define the appropriate duration of those tests. The authors assessed 1) how viability (the shell closure response to sodium chloride) compares with infectivity (ability to attach to a host fish and successfully metamorphose to the juvenile stage), and 2) the decline of viability and infectivity over time after glochidia were released from female mussels. Glochidia of 7 mussel species were isolated from females, placed in water, and subsampled daily for 2 d to 5 d. Viability, when ≥90%, was generally a good predictor of infectivity; however, when viability was <90%, infectivity was often disproportionately low, especially for glochidia collected near the end of the brooding period. Viability and infectivity declined more rapidly in natural water and sediment compared to reconstituted water. Following 24-h exposure to a toxicant (sodium chloride or copper), infectivity of the viable glochidia did not differ among concentrations of toxicants. The results indicate that viability is a valid proxy for infectivity and an ecologically relevant endpoint for standard toxicity tests with freshwater mussels for any test duration with control viability >90%.

Laboratory assessment of the antifouling potential of a soluble-matrix paint laced with the natural compound polygodial.

Polygodial is a potent and selective inhibitor of ascidian metamorphosis that shows promise for controlling fouling by ascidians in bivalve aquaculture. The current study examined the potency of, and associated effects of seawater exposure on, a rosin-based soluble-matrix paint laced with 0.08-160 ng polygodial g(-1) wet paint matrix. Paint-coated surfaces were soaked in seawater for 0, 2, 4 or 12 weeks prior to screening for antifouling activity using a bioassay based on the nuisance ascidian Ciona savignyi Herdman. Mortality was greater (mean 50% lethal concentration: 5 ± 2 ng g(-1); mean 75% lethal concentration: 17 ± 4 ng g(-1)) and metamorphosis was inhibited (mean 50% anti-metamorphic concentration: 2 ± 0.4 ng g(-1); mean 75% anti-metamorphic concentration: 15 ± 10 ng g(-1)) in C. savignyi larvae exposed to polygodial-laced soluble-matrix paints, relative to control paints without polygodial. Soaking in seawater prior to testing reduced the efficacy of the formulation up to nearly 12-fold, but even after soaking for 12 weeks paints laced with polygodial at 160 ng g(-1) wet paint matrix prevented ⩾90% of the larvae of C. savignyi from completing metamorphosis. The outcome of this experiment provides a positive first step in evaluating the suitability of polygodial-laced soluble-matrix paints for use in aquaculture.

Assessment of toxic interactions of heavy metals in multi-component mixtures using sea urchin embryo-larval bioassay.

The toxicities of copper, lead, zinc and cadmium ions and various concentrations of mixtures of them were studied using sea urchin (Strongylocentyotus intermedius) embryo-larval bioassay. Toxic unit analysis was used to determine type of joint action for each mixture combination (binary, ternary and quaternary). For the majority of the binary combinations, the interactions were of synergistic nature, but in ternary or quaternary mixtures, the joint action was mainly concentration additive, while antagonism was only observed for two mixtures (Cu+Pb and Zn+Cd) among all the 11 combinations. Two prevailing theoretical models: the concentration addition (CA) model and the independent action (IA) model were used to predict the mixture toxicities. The weak correlation obtained (R≃0.55) indicated that the hypotheses of mode of action involved in the two models to some extent failed to describe the behavior of the mixture system. Then a novel bio-concentration factor-based model was developed and was successful to predict the toxicities of mixtures, with an obtained R of 0.92. This model indicated that in a mixture system of heavy metals, the joint toxicity was mainly determined by the combined action of bio-concentrations of metals other than the simply similar (CA) or dissimilar (IA) modes of action of the mixture components.

Detection of Francisella tularensis in Alaskan mosquitoes (Diptera: Culicidae) and assessment of a laboratory model for transmission.

Tularemia is a zoonotic disease caused by the Category A bioterrorism agent Francisella tularensis. In Scandinavia, tularemia transmission by mosquitoes has been widely cited in the literature. We tested >2,500 mosquitoes captured in Alaska and found Francisella DNA in 30% of pooled samples. To examine the potential for transmission of Francisella by mosquitoes, we developed a mosquito model of Francisella infection. Larvae of Anopheles gambiae Giles and Aedes aegypti (L.) readily ingest F. tularensis but do not efficiently transfer infective doses of the bacterium to the pupal or adult stage. After a bloodmeal containing Francisella, adult female An. gambiae and Ae. aegypti retained detectable levels of Francisella DNA for 3 d, but when they took a second bloodmeal, the mammalian host was not infected. This study suggests that although Francisella DNA can be detected in a significant portion of wild-caught mosquitoes, transmission of Francisella is either very inefficient or is species dependent for the Francisella strain or the arthropod vector.