Right ventricular dysfunction in patients with COVID-19 pneumonitis whose lungs are mechanically ventilated: a multicentre prospective cohort study.

Cardiovascular complications due to COVID-19, such as right ventricular dysfunction, are common. The combination of acute respiratory distress syndrome, invasive mechanical ventilation, thromboembolic disease and direct myocardial injury creates conditions where right ventricular dysfunction is likely to occur. We undertook a prospective, multicentre cohort study in 10 Scottish intensive care units of patients with COVID-19 pneumonitis whose lungs were mechanically ventilated. Right ventricular dysfunction was defined as the presence of severe right ventricular dilation and interventricular septal flattening. To explore the role of myocardial injury, high-sensitivity troponin and N-terminal pro B-type natriuretic peptide plasma levels were measured in all patients. We recruited 121 patients and 118 (98%) underwent imaging. It was possible to determine the primary outcome in 112 (91%). Severe right ventricular dilation was present in 31 (28%), with interventricular septal flattening present in nine (8%). Right ventricular dysfunction (the combination of these two parameters) was present in seven (6%, 95%CI 3-13%). Thirty-day mortality was 86% in those with right ventricular dysfunction as compared with 45% in those without (p = 0.051). Patients with right ventricular dysfunction were more likely to have: pulmonary thromboembolism (p < 0.001); higher plateau airway pressure (p = 0.048); lower dynamic compliance (p = 0.031); higher plasma N-terminal pro B-type natriuretic peptide levels (p = 0.006); and raised plasma troponin levels (p = 0.048). Our results demonstrate a prevalence of right ventricular dysfunction of 6%, which was associated with increased mortality (86%). Associations were also observed between right ventricular dysfunction and aetiological domains of: acute respiratory distress syndrome; ventilation; thromboembolic disease; and direct myocardial injury, implying a complex multifactorial pathophysiology.

Association between anaesthetic technique and unplanned admission to intensive care after thoracic lung resection surgery: the second Association of Cardiothoracic Anaesthesia and Critical Care (ACTACC) National Audit.

Unplanned intensive care admission is a devastating complication of lung resection and is associated with significantly increased mortality. We carried out a two-year retrospective national multicentre cohort study to investigate the influence of anaesthetic and analgesic technique on the need for unplanned postoperative intensive care admission. All patients undergoing lung resection surgery in 16 thoracic surgical centres in the UK in the calendar years 2013 and 2014 were included. We defined critical care admission as the unplanned need for either tracheal intubation and mechanical ventilation or renal replacement therapy, and sought an association between mode of anaesthesia (total intravenous anaesthesia vs. volatile) and analgesic technique (epidural vs. paravertebral) and need for intensive care admission. A total of 253 out of 11,208 patients undergoing lung resection in the study period had an unplanned admission to intensive care in the postoperative period, giving an incidence of intensive care unit admission of 2.3% (95%CI 2.0-2.6%). Patients who had an unplanned admission to intensive care unit had a higher mortality (29.00% vs. 0.03%, p < 0.001), and hospital length of stay was increased (26 vs. 6 days, p < 0.001). Across univariate, complete case and multiple imputation (multivariate) models, there was a strong and significant effect of both anaesthetic and analgesic technique on the need for intensive care admission. Patients receiving total intravenous anaesthesia (OR 0.50 (95%CI 0.34-0.70)), and patients receiving epidural analgesia (OR 0.56 (95%CI 0.41-0.78)) were less likely to have an unplanned admission to intensive care after thoracic surgery. This large retrospective study suggests a significant effect of both anaesthetic and analgesic technique on outcome in patients undergoing lung resection. We must emphasise that the observed association does not directly imply causation, and suggest that well-conducted, large-scale randomised controlled trials are required to address these fundamental questions.

Efficacy and community effectiveness of larvivorous fish for dengue vector control.

OBJECTIVE: To evaluate the efficacy and community effectiveness of larvivorous fish for the control of dengue vectors and dengue transmission, when used as a single agent or in combination with other vector control methods. METHOD: Comprehensive literature search of published and grey literature using PubMed, EMBASE (DMDI), Web of Science, WHOLIS, WILEY, LILACS, GIFT, Cochrane Library, ELDIS, New York Academy of Medicine Grey Literature Report and Google. All results were checked for duplicates and examined for eligibility. Methodological quality of the studies was assessed using RoBANS. RESULTS: Thirteen articles were considered eligible for inclusion. Incorporating a wide range of interventions and outcome measures, three were efficacy studies and 10 assessed community effectiveness. None of the studies were randomised or cluster-randomised controlled trials. All three efficacy studies and seven community effectiveness studies investigated fish as a single agent. All efficacy studies reported elimination of Aedes larvae from treated containers, while community effectiveness studies reported reductions in immature vector stages, two of which also detected a continuous decline over 2 years. An impact on adult mosquitoes was shown in only two community effectiveness studies. Reductions in dengue cases following intervention were reported in two studies, but it was not possible to attribute this to the intervention. CONCLUSION: While the use of larvivorous fish as a single agent or in combination with other control measures could lead to reductions in immature vector stages, considerable limitations in all the studies restricted any conclusions with respect to the evaluation of community effectiveness. Evidence for the community effectiveness of larvivorous fish as a single agent remains minimal and cluster-randomised controlled studies that include the assessment of impact on dengue are recommended.

Critical care after lung resection: CALoR 1, a single-centre pilot study.

Lung resection is associated with significant perioperative morbidity, and a proportion of patients will require intensive care following surgery. We set out to characterise this population, assess their burden of disease and investigate the influence of anaesthetic and surgical techniques on their admission rate. Over a two-year period, 1169 patients underwent surgery, with 30 patients (2.6%) requiring unplanned intensive care. Patients requiring support had a higher mortality (0.2% vs 26.7%, p < 0.001). Logistic regression (following adjustment for Thoracoscore) revealed that an open surgical approach was associated with higher likelihood of admission (p = 0.025, odds ratio = 5.25). There was also a trend towards increased likelihood of admission in patients who received volatile anaesthesia (p = 0.061, odds ratio = 2.08). This topic has been selected for further investigation as part of the 2015 Association of Cardiothoracic Anaesthetists (ACTA) second national collaborative audit, with this study providing pilot data before a multi-centre study.

Dengue disease surveillance: an updated systematic literature review.

OBJECTIVES: To review the evidence for the application of tools for dengue outbreak prediction/detection and trend monitoring in passive and active disease surveillance systems in order to develop recommendations for endemic countries and identify important research needs. METHODS: This systematic literature review followed the protocol of a review from 2008, extending the systematic search from January 2007 to February 2013 on PubMed, EMBASE, CDSR, WHOLIS and Lilacs. Data reporting followed the PRISMA statement. The eligibility criteria comprised (i) population at risk of dengue, (ii) dengue disease surveillance, (iii) outcome of surveillance described and (iv) empirical data evaluated. The analysis classified studies based on the purpose of the surveillance programme. The main limitation of the review was expected publication bias. RESULTS: A total of 1116 papers were identified of which 36 articles were included in the review. Four cohort-based prospective studies calculated expansion factors demonstrating remarkable levels of underreporting in the surveillance systems. Several studies demonstrated that enhancement methods such as laboratory support, sentinel-based reporting and staff motivation contributed to improvements in dengue reporting. Additional improvements for passive surveillance systems are possible by incorporating simple data forms/entry/electronic-based reporting; defining clear system objectives; performing data analysis at the lowest possible level (e.g. district); seeking regular data feedback. Six studies showed that serotype changes were positively correlated with the number of reported cases or with dengue incidence, with lag times of up to 6 months. Three studies found that data on internet searches and event-based surveillance correlated well with the epidemic curve derived from surveillance data. CONCLUSIONS: Passive surveillance providing the baseline for outbreak alert should be strengthened and appropriate threshold levels for outbreak alerts investigated. Additional enhancement tools such as syndromic surveillance, laboratory support and motivation strategies can be added. Appropriate alert signals need to be identified and integrated into a risk assessment tool. Shifts in dengue serotypes/genotype or electronic event-based surveillance have also considerable potential as indicator in dengue surveillance. Further research on evidence-based response strategies and cost-effectiveness is needed.

Multi-scale analysis of the associations among egg, larval and pupal surveys and the presence and abundance of adult female Aedes aegypti (Stegomyia aegypti) in the city of Merida, Mexico.

Despite decades of research, there is still no agreement on which indices of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) presence and abundance better quantify entomological risk for dengue. This study reports the results of a multi-scale, cross-sectional entomological survey carried out in 1160 households in the city of Merida, Mexico to establish: (a) the correlation between levels of Ae. aegypti presence and abundance detected with aspirators and ovitraps; (b) which immature and egg indices correlate with the presence and abundance of Ae. aegypti females, and (c) the correlations amongst traditional Aedes indices and their modifications for pupae at the household level and within medium-sized geographic areas used for vector surveillance. Our analyses show that ovitrap positivity was significantly associated with indoor adult Ae. aegypti presence [odds ratio (OR) = 1.50; P = 0.03], that the presence of pupae is associated with adult presence at the household level (OR = 2.27; P = 0.001), that classic Aedes indices are informative only when they account for pupae, and that window screens provide a significant level of protection against peridomestic Ae. aegypti (OR = 0.59; P = 0.02). Results reinforce the potential of using both positive collections in outdoor ovitraps and the presence of pupae as sensitive indicators of indoor adult female presence.

Insecticide-treated bednets to control dengue vectors: preliminary evidence from a controlled trial in Haiti.

OBJECTIVES: Insecticide-treated bednets (ITNs) are effective in preventing nocturnally transmitted vector-borne diseases, but their effect on diurnally active dengue vectors has never been studied. We investigated the efficacy of ITNs in reducing Aedes aegypti populations and dengue transmission. METHODS: A cluster-randomized trial was carried out in Leogane, Haiti between July 2003 and July 2004. The study area (1017 houses) was divided into 18 sectors (clusters): nine received ITNs (Olyset(R) long-lasting insecticidal bednets) and nine were untreated controls. Entomological surveys [measuring Breteau (BI), house (HI), container (CI) and pupae per person (PPI) indices and oviposition activity] were undertaken at baseline and at 1 and 5 months post-intervention. All houses were georeferenced to enable spatial analysis. Control sectors received ITNs at 6 months, and a final entomological and attitudinal survey was undertaken at 12 months after baseline. Anti-dengue IgM seropositivity rates were measured at baseline and after 12 months. Efficacy of ITNs was assessed by WHO cone bioassays. RESULTS: At 1-month post-intervention, entomological indices fell in all sectors, with HI and BI in the bednet sectors reduced by 6.7 (95% CI -10.6, -2.7; P < 0.01) and 8.4 (95% CI -14.1, -2.6; P < 0.01) respectively. Moreover at 1 month, ovitraps in control sectors were significantly more likely to be positive than in bednet sectors (P < 0.01). By 5 months, all indices remained low and HI, CI and BI were also significantly lower than that of baseline in the control arm. Curiously, at 5 months, HI, CI and BI were lower in the control arm than that in the bednet arm. A final survey, 12 months after the initial baseline study (5 months after bednets had been given to all households) indicated that all indices were significantly lower than that at baseline (P < 0.001). Control houses located within 50 m of a bednet house had significantly lower CI (Z = -2.67, P = 0.008) and PPI (Z = -2.19, P = 0.028) at 1 month, an effect that extended to 100 m by 5 months (Z = -2.03, P = 0.042 and Z = -2.37, P = 0.018 respectively), suggesting a spill-over effect of the bednets. An IgM serosurvey showed a 15.3% decrease (95% CI 5.0-25.5%, P < 0.01) in the number of IgM-positive individuals from baseline to12 months later. CONCLUSIONS: Insecticide-treated bednets had an immediate effect on dengue vector populations after their introduction, and over the next 5-12 months, the presence of ITNs may have continued to affect vector populations and dengue transmission.

Does tick-borne relapsing fever have an animal reservoir in East Africa?

Tick-borne relapsing fevers (TBRF) are caused by infection with Borrelia spirochetes and transmitted to humans by ticks. All except East African TBRF, caused by Borrelia duttonii, are known zoonoses. This widespread, endemic and pathogenic infection has only been found in humans and the Ornithodoros sp. soft tick vectors. We investigated the role of domestic animals as possible reservoirs of infection in a TBRF endemic region. Tick infestations in households and pigpens were investigated in the villages near Mvumi hospital in central Tanzania. Blood from chickens and pigs was examined by PCR and flagellin gene sequencing was performed on any Borrelia sp. infections detected. A mark-recapture experiment investigated tick movement between pigpens and houses. The acceptability of chickens as tick hosts was also investigated. Tick infestation of the 122 houses investigated was high (47%). Pigpens also were tick infested (16%) and were more likely to be so if they were located close to tick infested households (p<0.001). PCR screening of peripheral blood found Borrelia infections in both chickens and pigs (11% and 8.9% respectively). Sequencing of a subset of positive samples revealed that the amplified Borrelia sp. flagellin gene fragments shared greatest homology with B. duttonii. In a mark-recapture experiment, ticks released in pigpens were recaptured inside human bedrooms. When offered chickens as hosts, over 20% of ticks fed. For the first time in East Africa, we record natural infections of Borrelia in domestic animals and show that tick populations may act as bridging vectors between animals and humans. These results, from villages where B. duttonii is already known to be prevalent and a major cause of illness in humans, and where it has been found at high levels in ticks, strongly support the case that it is a zoonosis. This increases understanding of the epidemiology and control of this important but neglected human disease.

A novel video-tracking system to quantify the behaviour of nocturnal mosquitoes attacking human hosts in the field.

Many vectors of malaria and other infections spend most of their adult life within human homes, the environment where they bloodfeed and rest, and where control has been most successful. Yet, knowledge of peri-domestic mosquito behaviour is limited, particularly how mosquitoes find and attack human hosts or how insecticides impact on behaviour. This is partly because technology for tracking mosquitoes in their natural habitats, traditional dwellings in disease-endemic countries, has never been available. We describe a sensing device that enables observation and recording of nocturnal mosquitoes attacking humans with or without a bed net, in the laboratory and in rural Africa. The device addresses requirements for sub-millimetre resolution over a 2.0 × 1.2 × 2.0 m volume while using minimum irradiance. Data processing strategies to extract individual mosquito trajectories and algorithms to describe behaviour during host/net interactions are introduced. Results from UK laboratory and Tanzanian field tests showed that Culex quinquefasciatus activity was higher and focused on the bed net roof when a human host was present, in colonized and wild populations. Both C. quinquefasciatus and Anopheles gambiae exhibited similar behavioural modes, with average flight velocities varying by less than 10%. The system offers considerable potential for investigations in vector biology and many other fields.

A newly identified tick-borne Borrelia species and relapsing fever in Tanzania.

Tick-borne relapsing fever caused by the spirochaete Borrelia duttonii is a common cause of serious illness in central Tanzania. Screening of Ornithodoros sp ticks from infested houses for the presence of B duttonii had detected a previously unidentified species of Borrelia. We investigated whether this species infected the human population in a central Tanzanian village, by use of blood slide examination and PCR. PCR was twice as sensitive in detection of infections, showing Borrelia sp in six (11%) of 54 children with fever, and in 13 (4%) of 307 otherwise healthy children. Genotyping Borrelia from 17 infections identified Borrelia duttonii and an unnamed species. Our findings show that the newly discovered species is a causal agent of tick-borne relapsing fever.

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae.

BACKGROUND: A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. METHODS: A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. RESULTS AND DISCUSSION: The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. CONCLUSION: The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.

Malaria and urbanization in sub-Saharan Africa.

There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.

Learning and memory in disease vectors.

All animals can learn to some extent and it should not be surprising to discover that important vectors can also be influenced by experience. The potential effect of memory on vector behaviour, particularly vectorial capacity, has barely been investigated. Yet, how a population of blood-feeding insects distributes between available resources has important epidemiological consequences. Several recent studies have shown that behaviour during oviposition site-selection, host location and even host choice can be influenced by the environment or by experience after eclosion. The significance of these studies and their consequences for epidemiology and control are considered here.

Cloning and characterization of a species-specific repetitive DNA sequence from Onchocerca armillata.

Two clones, pOA1 and pOA5, have been isolated from a genomic DNA library prepared from pools of Onchocerca armillata adults in the plasmid vector pUC12. In dot-blot hybridisations, these two clones do not cross-hybridise significantly with total genomic DNA from O. volvulus, O. gutturosa, O. ochengi, O. gibsoni, O. lienalis, bovine, human, Culicoides nubeculosus, Simulium species or Brugia pahangi, but do hybridise with as little as 100 pg of DNA from two separate geographic isolates of O. armillata. The sequence of pOA1 and pOA5 has been determined and found to contain a repetitive DNA sequence 147 bp in length. These clones can be used as specific and sensitive DNA probes for the identification of O. armillata capable of identifying a single L3 larva.

Morphometric differentiation of Onchocerca volvulus and O. ochengi infective larvae.

By microinjection of cryopreserved microfilariae (mf) into nulliparous flies, a comparison of the lengths of the infective larvae (L3) of Onchocerca volvulus and O. ochengi from the head of Simulium damnosum s.l. (presumed S. sirbanum) has been made. The suitability of S. sirbanum as a host was similar for both Onchocerca spp. The mean length +/- standard deviation of O. ochengi infective larvae measured in aqueous medium after storage of infected flies in liquid nitrogen was 762 +/- 63 microns (n = 39), significantly longer (P much less than 0.0001) than those of a savanna isolate of O. volvulus (676 +/- 56 microns, n = 26). Although the frequency distributions of the lengths of larvae of the 2 species overlapped, a critical value for discrimination of 719 microns applied to normally distributed populations with means and standard deviations of these samples would result in correct classification of 78% of true O. volvulus and 75% of true O. ochengi. A discriminant function analysis incorporating width measurements did not usefully improve the level of accuracy of discrimination. Larvae from flies stored in 70% ethanol and stained with acid haemalum were about 10% shorter, but O. ochengi infective larvae were still proportionately longer than those of O. volvulus (693 +/- 40 microns, n = 45 compared to 580 +/- 38 microns, n = 6, respectively). These data show that the infective L3 of O. volvulus and O. ochengi differ morphologically. Although the population length distributions overlap, by classifying larvae greater than 719 microns long as O. ochengi and those less than 719 microns long as O. volvulus a more accurate estimation of true O. volvulus infection rates in S. damnosum s.l. can be derived than is currently possible.

Evidence for memorized site-fidelity in Anopheles arabiensis.

A mark-recapture experiment was carried out in northern Tanzania to determine whether Anopheles arabiensis exhibits memory, by investigating if bloodfed individuals would return to either the location or the host where or on which they had obtained a previous bloodmeal, behaviours termed site-fidelity and host-fidelity respectively. Over 4300 mosquitoes were collected from 2 houses, marked with different fluorescent colours according to whether they were caught in cattle sheds, 'cattle-fed', or within human bednets, 'human-fed', at either location, then released from a third location. Over 17,000 mosquitoes were collected and examined over the next 8 days. In total, 1% of released mosquitoes were recaptured. Of these, 68% had returned to the house where they were first caught, demonstrating site-fidelity (P = 0.007). However, 86% of recaptured mosquitoes were caught on cattle regardless of where they were initially caught (P = 0.185). Bloodmeal identification showed that a high proportion of mosquitoes classed as human-fed contained bovine blood, thereby confounding the investigation into host-fidelity. Notably, the proportion of mosquitoes with mixed bloodmeals depended on the proximity of cattle and humans, with significantly higher proportions of mixed bloodmeals occurring when cattle and humans slept in close proximity. The effects of the observed behaviours on malaria epidemiology are discussed.

Identification of the vectors of lymphatic filariasis in the Lower Shire Valley, southern Malawi.

An investigation of lymphatic filariasis vectors in Malawi is reported. Anopheles funestus, A. arabiensis, and A. gambiae sensu stricto had high rates of filarial infection (2.2-3.1%) and carried infective larvae. Anopheles funestus was the predominant species collected (77.6%) and was the primary vector during the study period of April to May 2002.

Attraction and trapping of Aedes aegypti (Diptera: Culicidae) with host odors in the laboratory.

Volatile constituents of mouse odor were eluted from an adsorbent after collection within a closed-air system, and offered to host-seeking yellowfever mosquitoes, Aedes aegypti (L.), in the laboratory. Gas chromatographic analysis showed that individual mouse odors varied in blend, both quantitatively and qualitatively. In a 2-choice bioassay, sticky traps baited with individual mouse odors caught more female mosquitoes than controls (69% caught on baited traps) in all cases. When 2 traps baited with volatiles from the empty collection system were used, no preference occurred (46 and 54%), and fewer mosquitoes (22% of total) were caught overall than when a baited trap was present (46% of total). Equal numbers of male mosquitoes were caught on baited and control traps. Results demonstrated that Ae. aegypti can be attracted to entrained and eluted host odor without the addition of other attractants.

Assessing the relationship between vector indices and dengue transmission: a systematic review of the evidence.

BACKGROUND: Despite doubts about methods used and the association between vector density and dengue transmission, routine sampling of mosquito vector populations is common in dengue-endemic countries worldwide. This study examined the evidence from published studies for the existence of any quantitative relationship between vector indices and dengue cases. METHODOLOGY/PRINCIPAL FINDINGS: From a total of 1205 papers identified in database searches following Cochrane and PRISMA Group guidelines, 18 were included for review. Eligibility criteria included 3-month study duration and dengue case confirmation by WHO case definition and/or serology. A range of designs were seen, particularly in spatial sampling and analyses, and all but 3 were classed as weak study designs. Eleven of eighteen studies generated Stegomyia indices from combined larval and pupal data. Adult vector data were reported in only three studies. Of thirteen studies that investigated associations between vector indices and dengue cases, 4 reported positive correlations, 4 found no correlation and 5 reported ambiguous or inconclusive associations. Six out of 7 studies that measured Breteau Indices reported dengue transmission at levels below the currently accepted threshold of 5. CONCLUSIONS/SIGNIFICANCE: There was little evidence of quantifiable associations between vector indices and dengue transmission that could reliably be used for outbreak prediction. This review highlighted the need for standardized sampling protocols that adequately consider dengue spatial heterogeneity. Recommendations for more appropriately designed studies include: standardized study design to elucidate the relationship between vector abundance and dengue transmission; adult mosquito sampling should be routine; single values of Breteau or other indices are not reliable universal dengue transmission thresholds; better knowledge of vector ecology is required.