Guam Vector Management Strategic Plan (2017 – 2020)

Guam Division of Environmental Health, Department of Public Health and Social Services

Version 1

Acronyms and Abbreviations

BCDC – Bureau of Communicable Disease Control

BCHS – Bureau of Community Health Services

BCI – Bureau of Compliance and Investigation

CDC – U.S. Center for Disease Control and Prevention

CHIKV – Chikungunya virus

DART – Disease Arbovirus Research and Training (UC Davis, California)

DEH – Guam Division of Environmental Health

DENV – Dengue virus

DoD – US Department of Defense

DPH – Guam Division of Public Health

DPHSS – Guam Department of Public Health and Social Services

ELC – Epidemiological and Laboratory Capacity for Infectious Diseases Cooperative Agreement

EOC – Emergency Operations Center

EPA – U.S. Environmental Protection Agency

FSM – Federated States of Micronesia

GEPA – Guam EPA

GEPHL – Guam Environmental Public Health Laboratory

GPHL – Guam Public Health Laboratory

IPNC – Institut Pasteur New Caledonia

IRS – Indoors Residual Spraying

JBE – Japanese B Encephalitis

LF – Lymphatic Filariasis

MAC – Mosquito Advisory Committee

M&E – Monitoring and Evaluation

MSCP – Guam Mosquito Surveillance and Control Program

NPEHA – Northern Pacific Environmental Health Association

PHEP – Public Health Emergency Preparedness Program

PIHOA – Pacific Islands Health Officers Association

STIs – Sexually Transmitted Infections

WHO – World Health Organization

ZIKV – Zika virus

Acknowledgements

This document was developed through a consultation process which was initiated in February 2017, on Guam, U.S.A. Expertise in entomology, environmental health, and public health was solicited from Technical Officer, WHO, Malaria and Other Vectorborne & Parasitic Diseases Unit, Mr. Matthew Shortus, Principal Hospital Scientist, Medical Entomology, Pathology Westmead CPMR, Westmead, NSW Health Pathology/Marie Bashir Institute of Infectious Diseases and Biosecurity, Sydney Medical School, University of Sydney, Dr. Cameron Webb, Career Epidemiology Field Officer, CDC/OPHPR/D SLR, Dr. William Thane Hancock, Environmental Health Specialist, PIHOA, Dr. Greg Jennings, Laboratory Science Specialist, PIHOA, Mr. Remi Charlebois, Vector Ecologist, PIHOA, Ms. Eileen Jeffrey, the Guam DEH MSCP team, Mrs. Rosanna Rabago, Mrs. Michelle Lastimoza, Ms. Claire Baradi.

The author also acknowledges the continuous support of the PIHOA U.S-Affiliated Pacific Islands Regional Zika Emergency and Preparedness Project team, Mr. Kinsey McFadden (Project Manager), Ms. Jada Walton (Education Coordinator), Mrs. Matilda Ward (Administrative Assistant), and Mr. Sean Perez (Financial Officer).

Finally, the author acknowledges and thanks CDC Chief Research Entomologist, Dr. Harry Savage, for providing valuable entomological guidance.

By Elodie Vajda, Regional Entomologist, PIHOA.

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Table of Contents

Acronyms and Abbreviations…………………………………………………………...... ………………………………...…………i

Acknowledgements…………………………………………………………………………………………………………………………….…….ii

Table of Contents……………………………………………………………………………………………………………………………………..vi

Map of Guam…………………………………………………………………………………………………………………………………….…..vii

Executive Summary……………………………………………………………………………………………………………………………...viii

I. Introduction………………………………………………………………………………………………………………………………………….1

a. Country Profile………………………………………………………………………………………………………………………..1

i. Geography, Sociopolitics, and Demographics of Guam………………………………………………..1

ii. Guam Health System Analysis…………………………………………………………………………………….1

b. Background: Historical Mosquito-Borne Diseases and Vectors………………………………………………..2

II. Vector-Borne disease (VBD) Situation Analysis…………………………………………………………………………………….3

a. Epidemiological Risk Assessment…………………………………………………………………………………………….3

b. Current Vector Profile……………………………………………………………………………………………………………..3

c. Vector-Borne Disease Control Program Performance………………………………………………………………4

III. Overall Strategic Directions…………………………………………………………………………………………………………………4

a. Strategic Directions…………………………………………………………………………………………………………………4

b. Strategic Goals and Objectives………………………………………………………………………………………………..5

IV. Vector Surveillance…………………………………………………………………………………………………………………………….5

a. Routine Vector Surveillance Program………………………………………………………………...…………………..5

i. Adult Trapping and Surveillance………………………………………………………………………………….6

ii. Larval/Pupal Trapping and Surveillance……………………………………………….……………………..6

iii. Ovitrapping and Eggs Surveillance……………………………………………………………………………..6

iv. Pathogen Testing...... 7

b. Enhanced Vector Surveillance...... 7

i. Disease Outbreaks...... 7

ii. Exotic Vector Importation...... 8

V. Vector Control...... 9

a. Routine Vector Control Program...... 9

i. Source Reduction and Larval Control...... 9

ii. Community Mobilization...... 9

b. Enhanced Vector Control...... 10

i. Disease Outbreak...... 10

VI. Program Management...... 10

a. Planning and Implementation...... 10

b. Information Systems...... 11

c. Procurement and Supply Management System...... 11

d. Human Resources and Technical Assistance...... 11

e. Monitoring and Evaluation (M&E)...... 12

References...... 13

Annexes

Annex 1. Useful Links...... 14

Annex 2. Descriptions of Aedes, Anopheles, and Culex Species Currently Recorded Present on Guam....15

Annex 3.1. Vector Surveillance Protocol in Tier 1 Sites...... 18

Annex 3.2. Vector Surveillance Protocol in Tier 2 Sites...... 20

Annex 3.2.i. Additional Tier 2 Sites...... 21

Annex 3.3. Vector Surveillance Protocol in Tier 3 Sites...... 22

Annex 4.1. Adult Mosquito Trapping Protocol: BG-Sentinel 2 (Biogents, GmbH, Regensburg, Germany).23

Annex 4.2. Adult Mosquito Trapping Protocol: CDC Miniature Light Trap...... 26

Annex 4.3. Adult Mosquito Trapping Protocol: CDC Gravid Trap...... 28

Annex 4.4. Mosquito Egg Trapping Protocol: Ovitraps and Hay Infusion...... 30

Annex 4.4.i. Trap Sign/Label Template...... 32

Annex 4.5. Mosquito Larval/Pupal Surveillance: Conducting a Larval/Pupal Survey...... 33

Annex 4.6. Adult Mosquito Species Identification Guidelines...... 36

Annex 4.7. Locality and Det Labels Protocols and Templates...... 41

Annex 4.8. Pinning/Mounting of Adult Mosquito Specimens...... 46

Annex 5.1. BG-Sentinels, Ovitraps, Gravid Traps, Field Data Recording Template...... 53

Annex 5.2. Larval/Pupal Surveys Template...... 55

Annex 6.1. Catch-bag Removal (BG-Sentinels, CDC Light Traps, CDC Gravid Traps) and Transportation Protocol...... 58

Annex 6.2. Transport of Ovitrap Paddles to the Lab and Hatching of Eggs Protocol...... 60

Annex 7. Enhanced Mosquito Surveillance Activities for Levels 1, 2, and 3...... 62

Annex 8. Enhanced Surveillance Activities for Detection of Exotic Species Import on Guam...... 63

Annex 9. Routine Source Reduction Activities and Community Mobilization...... 64

Annex 10. Enhanced Vector Control for Disease Outbreaks and Mitigation of Human-Vector Contact....66

Annex 11. CalSurv Terms and Conditions...... 68

Annex 12. Monitoring and Evaluation for Mosquito Surveillance and Control...... 74

Map of Guam

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Executive Summary

In the effort to address the threat of Zika virus to Guam, the Guam Division of Environmental Health (DEH), Department of Public Health and Social Services (DPHSS) received entomological support from the Pacific Islands Health Officers Association (PIHOA) as of February 2017. In light of the recent opening of the Guam Environmental Public Health Laboratory (GEPHL), a core component of this support entailed the formulation of the Guam Vector Management Strategic Plan (2017 – 2020). This plan provides guidance for all aspects mosquito surveillance and control (i.e., mosquito collecting, species identification, pathogen testing, vector control, insecticide resistance monitoring, and public outreach).

Guam is free of endemic arboviral diseases, likely linked to the elimination of Aedes aegypti (Linnaeus) from Guam following World War II. Therefore, it is imperative that Guam keep Ae. aegypti from being re- introduced and re-established on the island. This requires vigilant mosquito surveillance and control at all ports of entry of the island, and timely response to reported cases of arboviral diseases, such as dengue, Zika, and chikungunya.

The Guam Vector Management Strategic Plan (2017 – 2020) is founded on sound and robust scientific evidence, as well as on best practices implemented by the US Center for Disease Control and Prevention (CDC), the World Health Organization (WHO), and the American Mosquito Control Association (AMCA), which are explained in the first part of this document. The guidelines for the activities surrounding mosquito control and surveillance are written in the form of protocols and are referred to as ‘Annexes’. These Annexes are to serve as guides to meet the minimum requirements for adequate and effective mosquito surveillance and control and may be further elaborated and/or adapted in alignment with the scientific evidence presented in the first part of this document.

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I. Introduction.

a. Country Profile.

i. Geography, Sociopolitics, and Demographics of Guam.

Guam is an unincorporated and organized territory of the United States. The island has been a U.S. non- self-governing territory since 1898, and its inhabitants have been U.S. citizens since 1950. According to the Central Intelligence Agency World Factbook, Guam counts a total population of 162,742 (July 2016 est.), with 94.5% of its population being urban, and rate of urbanization of 1.36% per year. Additionally, Guam is a highly strategic island for the U.S. military. The island harbors the U.S. Naval Base Guam (NBG) and the Andersen Air Force Base (AAFB). Thus, 6,000 military personnel reside on Guam, and an additional 5,000 U.S. Marines, along with 1,300 dependents are expected to arrive on Guam by 2022. The Department of the Navy is relocating U.S. Marine Corps forces to Guam from Okinawa, Japan.

Accordingly, Guam is a key partner for the U.S. and for the other U.S.-Affiliated Pacific Islands (American Samoa, Commonwealth of the Northern Mariana Islands, Federated States of Micronesia, Republic of Palau, and Republic of Marshall Islands). Guam’s location makes the island an economic and immigration hub as its bustling transportation system facilitates the import of goods, and a flourishing tourism industry.

Guam is the largest and southernmost of the 15 islands which compose the Mariana Islands archipelago in the Pacific Ocean. It is situated at 13.28o N, 144.47o E, and has an area of 210 km2 (338 mi2) Northern Guam is characterized by a forested coral line limestone plateau, while southern Guam harbors volcanic peaks blanketed with forest and grassland. The northern and central parts of Guam are home to dense populations. The climate is tropical marine; the weather is hot and extremely humid with very little seasonal temperature variation. The mean high temperature is 30o C (86o F) and the mean low temperature is 24o C (75.2o F). The months of January and February constitute the coldest months of the year, as temperatures during the night drop to the mid to low 20s ۡ C (68o F), and humidity levels are slightly lower. Annual precipitation reaches an average of 2,180 mm (86 in.). The dry season spans December through June, and the rainy season runs from July to November. Typhoon risk is highest during October and November, although they do constitute a year-round risk (Rueda et al., 2011).

ii. Guam Health System Analysis.

The Department of Public Health and Social Services (DPHSS) is divided into five divisions, including: the Division of Public Health (DPH) and the Division of Environmental Health (DEH). The DPHSS main office is in Mangilao. The DPH is further divided into five Bureaus. The Bureau of Community Health Services (BCHS) allocates federal grants to DPH programs such as non-communicable disease and health and wellness programs. The Bureau of Communicable Disease Control (BCDC) is responsible for human surveillance of communicable diseases. The BCDC reports and investigates diagnosed cases of communicable diseases in collaboration with health care providers and responders (see Annex 1 for investigation forms used by DPH). Clinical laboratory services are provided through the BCDC’s Guam Public Health Laboratory (GEPHL). The GEPHL tests and treats for tuberculosis, Hansen’s Disease, HIV/AIDS and other STIs, vaccine-preventable diseases, and viruses such as dengue virus (DENV) and Zika virus (ZIKV).

In the event of a public health emergency, such as an arboviral outbreak, Public Health Emergency Preparedness (PHEP) program is responsible for enhancing the public health response and control to mitigate and resolve the impacts of the disease outbreak. The PHEP is a federally-funded initiative since 2000. Funding also serves to build the capacity of the GPHL. DPH spearheads the coordination and development of the Guam Communicable Disease Reporting System.

DEH is governed by 22 legislative mandates and 2 executive orders. The division is further divided into 3 bureaus. The Bureau of Compliance and Investigation (BCI) is composed of 8 staff members, and administers 8 programs, including the Mosquito Surveillance and Control Program (MSCP). The MSCP is responsible for the surveillance and control of mosquitoes on Guam.

In the event of a public health emergency, the Government of Guam will activate the Emergency Operations Center (EOC), which has a partnership to address public health emergencies on Guam with U.S. Department of Defense (DoD).

b. Background: Historical Mosquito-Borne Diseases and Vectors.

According to the prewar government handbook for Guam, mosquito-borne pathogens were not a problem on the island. Reports of military personnel of that time state that although mosquitoes were present on the island, they were only a nuisance, and not a health threat. However, the situation shifted when the Americans reoccupied Guam. This reoccupation involved large movements of men and material from regions in the Pacific where mosquito-borne diseases were already endemic and prevalent.

Indeed, in 1944, Guam saw its first major arboviral and dengue outbreak (Rozeboom & Bridges, 1972). Since then, dengue has historically been Guam’s most troublesome endemic illness. Epidemics of dengue frequently occurred in late summers. Human cases of malaria were reported from Guam in 1966, 1969, 1975, and, 1980-1986 (Nowell, 1987). In December 1947, a Japanese B encephalitis (JBE) outbreak occurred on Guam (Nowell, 1987).

This 1947 JBE outbreak led to an extensive mosquito survey on island in 1948 by the 207th Malaria Survey Detachment, U.S. Army (reported by Yamaguti and LaCasse, 1950). This survey incriminated Culex annulirostris as the main vector of JBE, and was the first study to report the present of Anopheles (Nowell, 1987). Thus, the suspected mosquito vectors for the malaria cases of the ‘60s and ‘80s were not confirmed, although reported Anopheles subpictus and/or An. barbirostris group are plausible candidates. However, their vector potential has yet to be confirmed (Rueda, et al., 2011). Two additional Anopheles species, including a known malaria vector, An. sinensis, and An. lesteri, respectively, were recorded on Guam, but are currently not as common as other Anopheles on Guam (Rueda, et al., 2011). However, additional surveys of the Anopheles of Guam would help determine whether this is still accurate or not. An. lesteri was collected again from Guam by W. K. Reisen (unpublished, 2010), and the most recent catch of An. sinensis was by S. P. Wolf in 2007.

Following WWII and the Vietnam war, the U.S. military launched a campaign to eliminate Aedes aegypti. The intervention involved massive fogging of DDT in various high-risk areas of the island. This campaign appeared to be successful as the last Ae. aegypti collected on Guam was by Yamaguti and LaCasse (1950) in 1948 (Reisen, et al., 1972). However, after years of no reports of Ae. aegypti on Guam, in 1969, Andersen Air Force Base sent a single specimen of Ae. aegypti to the 5th Epidemiological Flight for identification. The catch of this specimen could have resulted from a reintroduction, an escape, or the

maintenance of a low-density population (Reisen, et al., 1972). Yet in 1971, Reisen et al (1972) collected two specimens of Ae. aegypti in Tumon. However, there have been no reports of Ae. aegypti since, and Ae. aegypti is considered eliminated from the island of Guam.

II. Vector Borne Disease (VBD) Situation Analysis.

a. Epidemiological Risk Assessment.

The main infectious diseases that threaten Guam’s population are malaria, dengue fever, chikungunya fever, Japanese encephalitis, yellow fever, filariasis, and more recently, Zika virus (Rozeboom & Bridges, 1972; Rueda, et al., 2011). Since the elimination of Ae. aegypti from Guam after WWII and the Vietnam war, the sporadic cases of dengue and malaria on Guam have thus far been non-endemic, as cases have all been returning travelers from endemic countries. Indeed, as of 2017, over the course of 11 years, 24 cases of travel-related dengue have been reported on Guam.

In this light, adequate and timely human surveillance ensures that a non-endemic case is reported and treated. While mosquito surveillance is an essential component of arboviral and parasitic management, human surveillance also plays a fundamental role in outbreak prevention. For arboviruses such as Zika, dengue, and chikungunya, and parasites such as Plasmodium, detection of cases in the local population is the most effective for detecting an arboviral or parasitic outbreak. Timely identification and response to such arboviruses and parasites is reliant on consistent and regular communication between healthcare providers, certain employers who are mandated to report communicable diseases, and DPHSS. In concordance to human surveillance conducted by DPH, the DEH must deploy the appropriate vector control measure(s) to minimize the likelihood of an endemic mosquito biting the infected individual and transmitting the pathogen to Guamanians, thereby initiating endemic transmission on the island.

b. Current Vector Profile.

The last island-wide mosquito collecting activities took place in the early 2000’s, but were not systematic, and were only temporary (Rueda, et al., 2011). As of September 2017, the MSCP surveils Guam’s only commercial port and a large disadvantaged community in Astumbo, Dededo.

Both the NBG and AAFB on Guam conduct local mosquito collecting, but these activities are confined to the base enclosures. Navy and Air Force personnel responsible for mosquito surveillance mostly deploy BG-Sentinel 2 traps based on reports of mosquito sightings and nuisance-biting. Typical sites include military housing and recreational areas. The Navy proceeds to sorting the males from females, and then from sorting the females to genus. All the females are then sent off-island for identification and pathogen testing. Paper copies of the results are sent back to the Navy and stored for record keeping. The Air Force personnel also separate the males from the females, but do not sort the females to genus. All females are sent to the mainland for identification and pathogen testing. The results are emailed back to the Air Force.

In planning for the Festival of the Pacific Arts (FestPac) 2016, the DEH wrote a draft mosquito surveillance and control plan in prevision of the arrival of visitors from islands in which Zika transmission was recorded. The plan highlighted activities to conduct during the event and after the event. DEH was particularly successful in their public awareness initiatives. No cases of Zika were reported on Guam following the FestPac event; however, two imported cases of dengue were reported. MSCP conducted a 200-meter

radius surveillance and distributed numerous educational materials. A press release was issued to encourage source reduction and bit prevention.

To date, the consensus is that there are 24 mosquito species on Guam, nine of which are Aedes species, five of which are Anopheles species, and seven of which are Culex species. Although considered eliminated from Guam, Aedes aegypti is still included due to its very close association with human activity, and the capacity of its eggs to be efficiently transported and to colonize urban environments. The proximity of many neighboring countries to Guam that do have Ae. aegypti present means that there is a very considerable risk of importing this species into Guam.

Refer to Annex 2 for a complete table of the Aedes, Anopheles, and Culex species recorded on Guam.

c. Vector Borne Disease Control Program Performance.

Surveillance of human populations is led by public health personnel of BCDC within DPH. DPH’s clinical lab has the capacity to test for DENV, ZIKV, and chikungunya (CHIKV) via Real Time Polymerase Chain Reaction (RT-PCR). The DPH clinical lab tests patients for these arboviruses should the patient present dengue-like, Zika-like, or chikungunya-like clinical symptoms, as described by the U.S. Centers of Disease Control and Prevention (CDC). The PCR method of testing enables the potential detection of ZIKV about 14 days post initial infection and detection of DENV about seven days post initial infection. As PCR only detects active viral infection, the clinical lab personnel may choose to send human specimens to Hawai’i State Lab for confirmatory testing via enzyme-linked immunosorbent assay (ELISA). All lab testing results produced through private clinics and providers must be forwarded to the GEPHL, so results may be transmitted to the facility, territorial epidemiologist, and the BCDC Administrator via facsimile or email.

Case investigation forms are utilized by DPH to determine travel history of patient and the information is conveyed to DEH for action. Mosquito surveillance, control, and public outreach are conducted of all suspected and positive VBD cases whether non-endemic, endemic, or of unknown origin within a 200- meter radius (656 feet) surveillance from the patient’s residence.

III. Overall Strategic Directions.

a. Strategic Direction.

The guiding direction for the Guam Vector Management Strategic Plan is to progressively (“step-wise”) implement an effective routine vector surveillance program with clear vector control response linkages to:

• The notification of key VBD cases in Guam; • The detection of the exotic vector Aedes aegypti in Guam; • The detection of an adult mosquito sample infected with key pathogens;

This will include:

• Progressive improvements in operational (field and laboratory) capacity to implement vector surveillance and response activities;

• Strengthened internal government and stakeholder oversight, collaboration, and information sharing in relation to VBDs, particularly between the DPH and the DEH; and • Improved capacity for planning, priority setting, and resource allocation to combat the VBD threat in Guam.

DPH and DEH will set and pursue Guam’s strategic goals, which should be aligned with the DPHSS’ Strategic Plan.

Noting the importance of continuing technical and financial assistance, DPHSS and DEH will maintain and strengthen key partnerships, including: Guam Environmental Protection Agency (GEPA), CDC, World Health Organization (WHO), Secretariat of the Pacific Community (SPC), Pacific Island Health Officers Association (PIHOA), and Northern Pacific Environmental Health Association (NPEHA).

b. Strategic Goals and Objectives.

By the end of 2020, DEH will have the capacity to detect and respond to all significant VBD threats in Guam through effective implementation of technically sound vector management strategies.

Under the strategy, DEH activities are grouped into three thematic operational areas, each with its own strategic objective:

1. Exotic vector incursions: Maintain the status of Guam as being free from Ae. aegypti. 2. Outbreak prevention and control: Respond aggressively to the detection of VBD cases and/or infected vectors and prevent explosive outbreaks of malaria, DENV, CHIKV, and ZIKV. 3. Routine Vector Management: Maintain effective routine year-round vector surveillance and source reduction activities.

IV. Vector Surveillance.

a. Routine Vector Surveillance Program.

Identified sites for routine vector surveillance on the island of Guam are divided into three Tiers to assist the MSCP team in targeting vector surveillance and control strategies:

Tier 1 – High-risk entry points for Ae. aegypti [e.g., A.B. Won Pat International Airport, and Port Authority (PAG)of Guam], as well as high risk transmission areas (e.g., schools, health facilities, and disadvantaged, densely populated, neighborhoods). Refer to Annex 3.1 for the protocol to conduct vector surveillance in Tier 1 sites. Refer to Annex 4.6 for mosquito species identification procedures.

Tier 2 – Moderate transmission risk areas, other built up areas with high population (e.g., tourism areas, wealthier suburbs). Refer to Annex 3.2 for the protocol to conduct vector surveillance in Tier 2 sites. Refer to Annex 3.2.i. for additional Tier 2 sites previously identified. Refer to Annex 4.6 for mosquito species identification procedures.

Tier 3 – Low density and natural environment settings; often rural and not densely populated (e.g., south of the island of Guam). Refer to Annex 3.3 for the protocol to conduct mosquito surveillance in Tier 3 sites. Refer to Annex 4.6 for mosquito species identification procedures.

i. Adult Trapping and Surveillance.

Adult mosquito sampling using various trapping tools provides information regarding vector distribution, population trends, transmission dynamics and risks, and assessment of the effects of vector control activities. Traps include BG-Sentinels, Gravid Traps, and CDC Miniature Light Traps. Adult surveillance is typically more sensitive than larval/pupal surveillance but is less reliant on the field technician’s proficiency and skill. As Ae. aegypti and Ae. albopictus usually cover small distances, assessing the presence of adult mosquitoes can be a robust indicator of proximity to cryptic larval habitats, particularly for Ae. albopictus. Moreover, recent evidence indicates that adult surveillance is more indicative of transmission risk levels than larval/pupal surveillance. Indeed, larval/pupal surveillance data does not correlate well with the abundance of the vector stage of the mosquito, i.e., the adult stage (Focks, 2004). Therefore, adult mosquito sampling is favored for mosquito surveillance.

ii. Larval/Pupal Trapping and Surveillance.

Larval/pupal surveillance, conducted as “container surveys,” are often utilized to assess the spatial distribution of the mosquito vector, and the diversity and availability of container (artificial and natural) habitats. Such surveys also allow collectors to identify the most/least productive egg-laying sites, as various studies across various geographic locations show that container preferences vary from one geographic region to the next (Burkot, et al., 2007). Similarly, pupal surveys specifically, further pinpoint which water-holding containers are most productive, i.e., containers which exhibit high pupal density indicate a “productive site” for mosquito larvae, since an elevated number of larvae survived to the pupal stage. Finally, the higher the number of locations collected from, the more representative the sample.

iii. Ovitrapping and Egg Surveillance.

Eggs are collected using ovitraps. These traps attract container-egg laying mosquito species, including, but not exclusive of, Ae. aegypti and Ae. albopictus. However, identification of species by examining the morphology of the eggs is impractical and thus, it is often required to rear the eggs to larvae (4th instar) or adult for species identification.

Ovitraps are cheap, easy to use, non-invasive, and sensitive. A sparse number of ovitraps may be sufficient to determine vector presence; fewer than 100 ovitraps can accurately determine the mosquito abundance in a large urban neighborhood. In a typical city, for example, one ovitraps is placed per block. However, egg surveillance is similar to adult surveillance in that ovitraps do not supply information about the availability and type of larval habitat.

Moreover, caution must be taken when assessing ovitraps data, as ovitraps compete with naturally occurring larval habitats and the estimates from oviposition surveys may not actually reflect the abundance of gravid females. This is particularly important in the instance following source reduction efforts; gravid females find less water-holding containers and thus, more gravid females lay their eggs in the deployed ovitraps, further confounding the evaluation of source reduction efforts.

iv. Pathogen Testing.

As it is in the scope of the GEPHL to conduct routine viral ribosomal nucleic acid (RNA) testing of mosquitoes, it is relevant to discuss the calculation of adult infection rates.

Efficient adult mosquito trapping with BG-Sentinel traps and gravid traps has enabled the estimation of adult mosquito abundance and the tracking of infected mosquitoes. This may help in identifying local entomological infection rate thresholds for human disease risk for DENV, CHIKV, ZIKV, and yellow fever virus (YFV) transmission (CDC, 2013). The infection rate in a vector mosquito population is an estimate of the prevalence of an arbovirus-infected mosquito in the population.

Adult mosquitoes must be collected fresh for arboviral RNA testing. The recommended pool (a collection of a single mosquito species from the same site/collection container) of mosquitoes is 50 females; however, GEPHL may test pools of 100 females. Testing pools of 100 females will allow DEH to reduce the number of pools tested by half when there are staff limitations.

To calculate the infection index, MSCP shall utilize the Minimum Infection Rate (MIR):

Infection Index Formula Description/Assumptions MIR No. of arbovirus-positive pools/Total no. of Expressed as the no. of mosquitoes tested (not the number of infected/1,000 tested pools tested) (assumption: infection rates generally low)

The calculation of the MIR provides a quantitative basis for comparison to evaluate spatial and temporal changes in infection rates. Additionally, the MIR allows for different pool numbers and pool size without losing their comparability.

The accuracy of this index is dependent on the number of trap nights used to estimate abundance, and the number of specimens tested for viral RNA presence. However, adult mosquito infection rates are not used to predict outbreaks in DENV, CHIKV, ZIKV, and YFV surveillance programs. This is because there is currently limited data on infection rates and prevalence of human infections. Previous data acquired in DENV surveillance programs suggest that in certain instances, a rise in mosquito infection rates preceded dengue outbreaks or increased DENV transmission (Chow, et al., 1998; Mendez, et al., 2006). Thus, DEH must routinely calculate adult infection rates, should Guam mosquitoes test positive for arboviral RNA.

b. Enhanced Vector Surveillance.

i. Disease Outbreaks.

Enhanced mosquito surveillance must be initiated in the event of the following three scenarios, rated at three incremental risk levels:

Level 1 – A returning traveler with a confirmed DENV, ZIKV, or CHIKV infection (or other mosquito- borne illnesses such as malaria or yellow fever); likely a non-endemic case because of recent travel history.

Level 2 – A confirmed case of DENV, ZIKV, or CHIKV (or other arboviral/parasitic mosquito infection), that may potentially be locally acquired, as travel history is non-existent or does not align with the timing and results of infection diagnostic.

Level 3 – Outbreak: multiple (n=2+) confirmed cases of DENV, ZIKV, CHIKV, or other arboviral/parasitic mosquito infection, outside of a same family unit (which may have previously traveled together to a region with endemic arboviral/parasitic infections). This is strongly indicative of local pathogen transmission. A public health emergency should be declared if (1) local transmission is confirmed, and (2) local transmission is confirmed and Ae. aegypti is detected.

Specific activities and objectives of enhanced mosquito surveillance are outlined in Annex 7. Such activities are to be conducted in coordination with DPH and health professionals. The DEH’s sites of enhanced surveillance should be guided by the epidemiological data disclosed by the DPH to the DEH (see Box 1). Collaborative response measures between the DEH and DPH will ensure that the DEH MSCP uses its time and resources efficiently.

Box 1. Diagnostic information disclosed by DPH to DEH.

It is important that the DPH readily communicates the relevant diagnostic information to DEH. If a patient tests positive for ZIKV, DENV, or CHIKV via RT-PCR (IgM), then the patient is in its viremic stage and is infectious, i.e., if a local mosquito vector species bites the viremic individual, the local mosquito may acquire the virus and transmit it to another individual, thereby initiating local arboviral transmission. The infectious stage lasts about 14 days. Thus, it is crucial that DEH be informed by the DPH of this diagnostic, so that DEH may immediately deploy enhanced vector surveillance and control. However, if a patient tests negative for ZIKV, DENV, or CHIKV via RT-PCR, but positive via ELISA (IgG), then the DPH should only inform the DEH about this case if the DPH-lead patient investigation suggests local acquisition of the virus as indicated by the patient’s recent travel history. ii. Exotic Vector Importation.

Mosquito baseline data for Guam strongly suggest that the main vector of diseases presently on Guam is Ae. albopictus. Contrasting to most of its neighboring islands, Guam does not harbor Ae. aegypti. However, Guam is highly vulnerable to the re-introduction of Ae. aegypti, due to frequent traveling between Guam and its neighboring islands. Other vectors of the Pacific include Ae. hensilli (currently recorded in FSM and Republic of Palau), and Ae. polynesiensis (American Samoa). These three vectors have been responsible for various arboviral outbreaks in the Pacific such as, ZIKV, DENV, and lymphatic filariasis (LF) outbreaks. Thus, DEH must ensure the (re) introduction of these vector species is prevented and if detected, its spread limited and suppressed.

Thus, DEH should be prepared to respond in the event of the following three scenarios:

Scenario 1 – Ae. aegypti is detected inside military enclosures,

Scenario 2 – Ae. aegypti is detected outside military enclosures, and

Scenario 3 – Other exotic vector species is detected, such as Ae. hensilli, and/or Ae. polynesiensis outside military enclosures.

Refer to Annex 8 for the activities of enhanced mosquito surveillance in the event of either of the 3 scenarios mentioned above. The overall objective of these activities is to provide sufficient data to determine whether the exotic vector species detected is incidental (i.e., an ‘escape’), or established on Guam (see Annex 8).

V. Vector Control.

a. Routine Vector Control Program.

i. Source Reduction and Larval Control.

Environmental management, including integrated vector management, to prevent and/or mitigate the proliferation of mosquito populations on Guam is the pillar of a vector management program. Environmental management is primarily done in the form of source reduction, i.e., removing artificial and natural water-holding containers to limit mosquito egg-laying. Thus, routine source reduction is a form of larval control that must be conducted in concordance with routine mosquito surveillance, i.e., adult mosquito trapping and routine larval/pupal surveys. Conducting routine mosquito surveillance and routine mosquito control simultaneously will indicate effectiveness of vector control strategies employed.

Targeted sites for source reduction are prioritized and selected based on epidemiological data, such as:

(1) The identification of elevated numbers of mosquitoes. Relative increases in mosquito numbers (i.e., mosquito abundance) indicates the potential for an outbreak is higher than when mosquito numbers are lower. Such numbers are to be determined by MSCP as routine mosquito surveillance data is accumulated.

(2) Historical data, such as sites of past outbreaks (if applicable), and sites which consistently yield high numbers of mosquitoes as reported through DEH’s routine mosquito surveillance program. Such sites may indicate where an outbreak is most likely to start and expand.

(3) The identification of key water-holding containers, such as tires.

Source reduction must be conducted year-round (including during the dry season), and not only as a responsive measure. This means that source reduction targets key breeding sites in advance and during transmission seasons on Guam. This reduces the opportunity for breeding between outbreaks, thereby reducing the scale and frequency of arboviral outbreaks.

Larval control also includes the use of larviciding. However, on Guam, routine larviciding should be avoided, especially when there is no arboviral transmission island and/or no confirmed case of VBD.

Refer to Annex 9 for source reduction activities for a routine vector control program.

ii. Community Mobilization.

Community engagement is crucial to the sustainability and thoroughness of source reduction for vector control. For the general population to get engaged in mosquito control and source reduction, the community must be educated on the matter. It is the responsibility of the DEH to educate the population about the health risks posed by mosquitoes and how to help reduce mosquito populations around the

community. Information regarding mosquitoes and source reduction should be readily available to the public through social media, government website, radio announcements, and educational posters concentrated in areas harboring vulnerable populations (e.g., health clinics, DPHSS building, and schools).

Refer to Annex 9 for activities to promote community mobilization along with DEH source reduction activities.

b. Enhanced Vector Control.

i. Disease Outbreak.

In the event of a mosquito-borne disease (MBD) outbreak on Guam, vector control activities must immediately be deployed and closely monitored through mosquito surveillance to determine effectiveness of vector control strategies. The objective of vector control measures during an outbreak is to suppress the inferred mosquito vector population in and around outbreak location on Guam to reduce the risk of further arboviral transmission within the human population. Enhanced vector control must also be deployed in high- and moderate-risk sites (e.g., schools, health facilities) near outbreak location.

Enhanced vector control may implicate the use of chemical control such as larvicides and indoor residual spraying (IRS). Chemical control should only be used if there is the capacity to ensure adequate coverage to reduce transmission. Thus, DEH is responsible for carefully monitoring the effect of chemical control on the local mosquito population. This involves insecticide resistance testing by the GEPHL.

Finally, as described above, community mobilization is key to the effectiveness of enhanced vector control activities. Activities outlined in Annex 9 should be reinforced.

Refer to Annex 10 for enhanced vector control activities and for strategies to mitigate human-mosquito contact. Refer to Annex 1 for important links to U.S. EPA and CDC information pages on chemical and biological control of mosquitoes.

VI. Program Management.

a. Planning and Implementation.

The Mosquito Advisory Council (MAC) is to serve as the steering committee for vector management and control on Guam. The establishment of the MAC is part of Guam DEH’s Five Year Strategic Plan (FY 2015 – FY 2019), as Target 3, Aim 4, under the Animal and Vector Control Programs: mosquito, rodent, livestock, poultry, and fly.

Memberships include a variety of stakeholders, such as: AAFB Environmental Health Office; Department of Agriculture; Department of Parks and Recreation; Dr. Thane Hancock, CDC/PIHOA; Dr. Robert Haddock, DPH; Dr. Suzanne Kaneshiro, DPH; Guam Customs and Quarantine Agency; GEPA; Guam Hotel and Restaurant Association; Guam Homeland Security/Office of Civil Defense; Guam Veterans Cemetery; Guam Army National Guard/Environmental Programs; Guam Visitors Bureau; Senator Dennis G. Rodriguez, Jr.; Mayor’s Council of Guam; Port Authority of Guam; University of Guam/College of Natural and Applied Sciences (CNAS); U.S. Coast Guard Sector Guam; U.S. Department of Interior/National Park Service; U.S. Naval Hospital Guam/Environmental and Preventive Medicine Unit.

The MAC recognizes the importance of a collaborative and multi-sectoral approach to address MBD on Guam.

b. Information Systems.

The systematic collection and analysis of mosquito data is key to Guam’s vector management program. Data that is processed appropriately and regularly will allow for better vector monitoring and will serve as robust historical data. Historical data is important for a targeted vector control approach as it will indicate which sites on the island are more vulnerable to MBD outbreaks.

Therefore, mosquito data must be entered electronically onto a database on a regular basis, at least once per week. The database selected to serve as the GEPHL’s database should meet the following requirements: (1) be user friendly; (2) allow all Pacific vector mosquito species and non-vector mosquito species to be entered; (3) map various types of field data including trap types, date of sampling, location name of sampling site, etc.; (4) be protected from data tampering; and (5) be easily accessible to authorized organizations and institutions, such as WHO, CDC, and Pasteur Institute New Caledonia (PINC).

Moreover, as DEH is recipient of the CDC Epidemiological and Laboratory Capacity (ELC) Grant, MSCP is required to submit quarterly reports of mosquito data to MosquitoNET (see Annex 1 for link). The main purpose of MosquitoNET is to collect and compile data recording both Aedes aegypti and Aedes albopictus across the United States and the USAPIs. The data will be mapped out, and will be shared on a CDC website which is accessible to the public. Thus, MosquitoNET cannot serve as the only database the GEPHL utilizes to record and store its mosquito data: (1) MosquitoNET does not provide an interface for data analysis; and (2) MosquitoNET does not enable the GEPHL and other Pacific organizations/institutions (e.g., WHO, PINC) to share mosquito data.

As of September 2017, GEPHL is utilizing CalSurv, a database that is operated by University of California, Davis’ Davis Arbovirus Research and Training (DART) group (see Annex 1). Refer to Annex 11 for the terms and scope of CalSurv.

c. Procurement and Supply Management System.

As of September 2017, Global Scientific Solutions for Health (GSSHealth) through contract with PIHOA is developing a budget estimation tool and inventory management system for MSCP. The estimated completion date is scheduled for December 2017 and will be submitted to DEH for review and adoption.

d. Human Resources and Technical Assistance.

For effective vector management on Guam, DEH must ensure that it possesses the adequate human and other resources capacity. The table below outlines some of the key staff requirements and skill necessary for the long-term sustainability of Guam’s vector management program. GSSHealth has formulated a comprehensive staffing and training plan for the GEPHL. The estimated completion date is scheduled for December 2017 and will be submitted to DEH for review and adoption.

Table 1. Minimum necessary staff needs and skill for effective mosquito surveillance and control. Staff Requirements Skill Laboratory Director Oversees facility maintenance and security, laboratory budget, and laboratory operations Public Health Laboratory Administrator Supervises daily laboratory operations, conducts inventory management, develops and manages standard operation procedures Medical Entomologist Conducts mosquito identification, rearing, pathogen-testing, and insecticidal resistance assays. Environmental Public Health Officer Data entering, management, mapping, analysis, and reporting. Environmental Technician Conducts weekly trapping of mosquitoes.

MSCP will also work with partner agencies such as, CDC, WHO, PIHOA, and SPC to build technical and human resource capacity for GEPHL and its development as a regional laboratory.

e. Monitoring and Evaluation (M&E).

Monitoring and evaluation enables program implementers and managers to understand the cause-and- effect associations between implementation and impact. This highlights gaps and challenges, and are utilized to enhance vector management program strategy. For example, the absence of a specific outcome is often traced back to the absence of an input (i.e., activity). Refer to Annex 12 for M&E framework for vector surveillance and vector control. Additionally, in the absence of a structured accreditation program or standard for environmental public health laboratories, DEH is reviewing the possibility of becoming ISO 9001 or ISO 15189 compliant or accredited. Such accreditation may enhance commercial opportunities and provide greater recognition of the quality standards of GEPHL as a regional center for excellence.

References. American Mosquito Control Association. 2017. Best practices for integrates mosquito management: a focused update.

Center for Disease Control and Prevention. 2013. West Nile Virus in the United States: guidelines for surveillance, prevention, and control.

Center for Disease Control and Prevention. 2016. Guidelines for Aedes aegypti and Aedes albopictus surveillance and insecticide resistance testing in the United States. Version 2.

Focks, D.A. 2004. A review of entomological sampling methods and indicators for dengue vectors. UNICEF/UNDP/World Bank/WHO.

Guam Department of Public Health and Social Services. 2017. Division of Environmental Health. http://www.dphss.guam.gov/content/division-environmental-health

Haddock, R. L. 2010. A history of health on Guam. Crushers Football (Soccer) Club, Hagåtña , GU.

The Central Intelligence Agency World Factbook. 2017. World Factobook. https://www.cia.gov/library/publications/the-world-factbook/

Nowell, W.R. 1987. Vector introduction and malaria infection on Guam. J. Am. Mosq. Control Assoc. 3: 259 – 265.

Reisen, W. K., J.P. Burns, and R.G. Basio. 1972. A mosquito survey of Guam, Marianas Islands with notes on the vector borne disease potential. J. Med. Ent. 9: 319-324.

Rozeboom, L. E., and J. R. Bridges. 1972. Relative population densities of Aedes albopictus and A. guamensis on Guam. Bull. Wld. Health. Org. 46: 477 – 483.

Rueda, L. M., J. E. Pecor, W. K. Reeves, S. P. Wolf, P. V. Nunn, R. Y. Rabago, T. L. Gutierrez, and M. Debboun. 2011. Mosquitoes of Guam and the Northern Marianas: distribution, checklists, and notes on mosquito- borne pathogens. US Army Med. Dep. J. 17-28.

State of Queensland Health (Queensland Health). 2014. Queensland chikungunya management plan 2014-2019.

State of Queensland Health (Queensland Health). 2015. Queensland dengue management plan 20152020.

World Health Organization. 2003. Guidelines for dengue surveillance and mosquito control.

World Health Organization. 2016. Vector surveillance and control at ports, airports, and ground crossings.

Annex 1. Useful Links.

1. Disease investigations forms and other relevant documentation used by BCI: https://www.dphss.guam.gov/content/laboratory-services

2. Mosquito Species of Guam Identification Key (by L. Guillaumot, Institut Pasteur of New Caledonia): http://www.institutpasteur.nc/mosquitoes-and-disease-transmission/#keys_and_illustrations

3. Mosquito Species Associated with Dengue Transmission Pictorial Key: L. M., Rueda. (2004) Pictorial keys for identification of mosquitoes (Diptera: Culicidae) associated with Dengue Virus transmission, Zootaxa, 586: 1 – 60. http://www.dtic.mil/dtic/tr/fulltext/u2/a511584.pdf

4. MosquitoNET portal: https://www.cdc.gov/zika/vector/for-professionals.html

5. Questions about MosquitoNET, contact: [email protected].

6. CalSurv Gateway: https://gateway.calsurv.org/

7. CalSurv database webinar: https://www.youtube.com/watch?v=CnjDToSYC7E

8. DART group (UC Davis, CA): http://barkerlab.ucdavis.edu/dart/

9. WHO Handbook for vector management and surveillance at ports of entry: http://www.who.int/ihr/publications/9789241549592/en/

10. EPA Larval Control options: https://www.epa.gov/mosquitocontrol/controlling-mosquitoes-larval- stage

11. CDC Mosquito Control pg: https://www.cdc.gov/westnile/faq/mosquitocontrol.html

12. Online interactive WRBU key: http://www.wrbu.org/VecID_MQ.html

Annex 2. Descriptions of Aedes, Anopheles, and Culex species currently recorded present on Guam.

Species Medical relevance Egg-laying Sites Behavior/Ecology Recommended Trap Type Aedes aegypti ZIKV, DENV, CHIKV, Artificial & rarely Tropical, subtropical, BG-Sentinel Traps, YF, LF, JBE. natural water-holding temperate climates. Gravid traps, Ovitraps, containers near Flight range ~ 200-400 Mechanical human dwellings. m. Daytime feeder; larval/pupal (Artificial water- prefers to feed collecting, Aspirator. holding containers: indoors. Eggs not affected by desiccation-resistant. rainfall patterns). Multivoltine. Aedes albopictus ZIKV, DENV, CHIKV, Artificial & natural Tropical, subtropical, BG-Sentinel Traps, YF, JBE. water-holding temperate climates. Gravid traps, Ovitraps, containers. Optimal dvpt temp: Mechanical 25-30 degrees C. larval/pupal Flight range ~ 200 m. collecting, Aspirator. Daytime feeder. Eggs desiccation-resistant. Multivoltine. Aedes guamensis None reported. Mostly collected in Pop. abundance Mechanical natural containers declined because of larval/pupal collecting (coconut shells, tree competition w/ Ae. (BG-Sentinel, Gravid, holes, etc.), but also albopictus. Ovitraps may also found in artificial work) containers. Aedes neopandani None reported. Larvae commonly Adults occur near Mechanical collected from heavily wooded areas. larval/pupal Pandanus, taro leaf Aggressive biters. collecting, Aspirator, axils (BG-Sentinel, Gravid, Ovitraps may also work) Aedes oakleyi None reported. Mostly natural water- BG-Sentinel Traps, holding containers, Gravid traps, Ovitraps, but also in artificial Mechanical containers. larval/pupal collecting. Aedes pandani None reported, but Mostly natural water- Aggressive, daytime BG-Sentinel Traps, AFB (Guam) tests for holding containers biters. Aspirator, Gravid DENV, CHIKV, and (Pandanus axils, taro traps, Ovitraps, Flavivirus fields, coconut shells) Mechanical but also in artificial larval/pupal containers. collecting. Aedes rotanus None reported. Larvae mostly Severe biters. BG-Sentinel Traps, collected in Pandanus Gravid traps, Ovitraps, leaf axils; less Mechanical frequently in tree larval/pupal holes, and artificial collecting, Aspirator. containers. Aedes saipanensis None reported. Larvae collected in BG-Sentinel Traps, Pandanus leaf axils & Gravid traps, Ovitraps, artificial water- Mechanical holding containers. larval/pupal collecting. Aedes vexans Lab inoculation w/ JBE Larvae collected in Zoophilic, i.e. strong BG-Sentinel Traps, ground pools, tendency to feed on Aspirator, CDC Light swampy, marshy mammals; most Traps, Mechanical areas, tree holes, important sp. in

flooded grasses, taro floodwater; lay eggs in larval/pupal fields, cisterns. damp grass; strong collecting. flyers; tend to hide in vegetation. Anopheles Potential vector for Larvae found in deep, Zoophilic (esp. CDC Light Traps, barbirostris spp. malaria; associated w/ fresh, slow moving bovids); known to Mechanical group LF transmission water; sunlit to occupy highland & larval/pupal moderately shaded coastal areas; swamp collecting. areas; can be found in breeder; tolerant to ground pools w/ high high levels of organic concentrations of pollution; generally animal dung. outdoor feeders but adults may rest indoors; nighttime biters. Anopheles indefinitus Poor vector potential. Larvae collected from Evening/night feeder. CDC Light Traps, temporary ground Mechanical pools. larval/pupal collecting. Anopheles litoralis vivax & falciparum Larvae have been Salt-water/brackish Night catching via malaria. collected from breeder; aspiration around artificial containers. evening/night feeder. homes; CDC Light Trap around & inside human shelters, Mechanical larval/pupal collecting. Anopheles subpictus vivax & falciparum Larvae usually Strongly zoophilic; Night catching via malaria; although collected from muddy rarely bites humans. aspiration around level of vector pools along houses, homes; CDC Light potential is uncertain. barrow pits, hoof traps around & inside prints, & artificial human shelters, containers. Mechanical larval/pupal collecting. Anopheles vagus Malaria; vector Larvae found in range Generally zoophilic Night catching via potential of habitats, e.g. but some studies have aspiration around geographically muddy or clear pools, shown its tendency to homes; CDC Light variable. beach, fields, w/ be anthropophilic. traps around & inside preference for non- human shelters, polluted water. Mechanical larval/pupal collecting. Culex annulirostris Associated with JBE. Larvae collected in Zoophilic. CDC Light Traps, BG- marianae ground pools, esp. Sentinels, Mechanical with mud bottoms, larval/pupal artificial containers, collecting. swamps, agricultural fields. Culex fuscocephalus None reported Marshes. Feed primarily on CDC Light Traps, BG- (potential for JBE). non-human Sentinels, Mechanical mammalian hosts. larval/pupal collecting. Culex litoralis None reported. Larvae found in Feed primarily on Mechanical brackish water of non-human larval/pupal coral rock holes, mammalian hosts. collecting. artificial containers;

sp. rare during dry season. Culex LF. Larvae favor dirty and Adults nocturnal, CDC Light Traps, BG- quinquefasciatus polluted water: mainly ornithophilic Sentinels, Mechanical puddles, ditches, (prefer to bite birds), larval/pupal drains, sceptic tanks; but if no birds, will collecting. may co-occur with Ae. feed on humans. aegypti. Culex sitiens LF. Larvae found in Adults nocturnal; CDC Light Traps, BG- brackish water of aggressive biters. Sentinels, Mechanical coral rock holes, larval/pupal artificial containers; collecting. dirty water with trash (even in full sunlight); sometimes associated with Cx. litoralis; most common during wet season. Culex JBE Larvae collected in Zoophilic; nocturnal; CDC Light Traps, BG- tritaeniorhynchus ground pools, esp. very similar to Cx. Sentinels, Mechanical with mud bottoms, annulirostris marianae larval/pupal artificial containers, in appearance. collecting. swamps, agricultural fields. Lutzia fuscanus None reported Larvae collected from Natural predators of CDC Light Traps, BG- (potential for JBE). marshes, slow water Aedes larvae (e.g. Sentinels, Mechanical drains, large artificial aegypti, An. subpictus, larval/pupal water jars, cement Cx. tritaeniorhynchus); collecting. mud tanks, water pits. known to co-occur with larvae Cx. sitiens, Ae. albopictus, An. sinensis, Cx. quinquefasciatus, Cx. vagans; Larvae feeds on Ae. aegypti larvae.

Annex 3.1. Vector Surveillance Protocol in Tier 1 Sites.

Weekly (i.e., minimum 1x to 2x per week) mosquito surveillance should be conducted across the selected Tier 1 sites (i.e., ‘Sentinel Sites’). DEH should select six to eight Tier 1 sites, only from the North of the island. The North is more densely populated and more urbanized than the South, and thus, transmission risks are substantially higher in the North than in the South of the island. Collecting sites are defined as areas which cover about a 200-meter radius. However, 200-meter radius sites which have large roads (e.g., typical four-lane roads), such as Marine Corps Drive, should be subdivided into separate sites, as mosquito populations are known to vary substantially from large road separations.

The recommended Tier 1 sites are as follows:

1. A.B. Won Pat International Airport (ABWPIA) in Tamuning, 2. PAG in Piti, 3. Two sites in Astumbo area (middle school, elementary school) in Dededo, 4. Guam Memorial Hospital Authority (GMHA) in Tamuning, and 5. Guam Regional Medical City (GRMC) in Dededo.

Ports of entry are always considered to be high-risk areas because (a) they are an entry way for exotic mosquitoes onto Guam, and (b) they are the first place an infected traveler might expose him/herself to local mosquito populations.

It is advised that BG-Sentinels be used across all selected Tier 1 sites. However, resource limitations may not allow for all Tier 1 sites to be surveilled with BG-Sentinels, as these traps need their batteries to be replaced at the latest, every 48 hours. Table 1 outlines a proposed schedule for the Tier 1 sites listed above. Refer to the trapping protocols (Annex 4.1 to Annex 4.5) for properly using traps for adult, egg, and larval/pupal surveillance. Note that for Tier 1 vector surveillance, traps are to be left permanently in their designated areas.

Field data must be recorded during every collecting trip. Field data recording templates were written with PIHOA entomologist and DEH staff (Annex 5.1, Annex 5.2). The field data sheets ensure that all necessary information be collected to build important baseline data and to report results to CDC’s MosquitoNET. Field data sheets should be digitized for safekeeping.

Transport of mosquito specimens from the BG-Sentinels and from ovitraps in the field back to the laboratory should abide to Annex 6.1 and to Annex 6.2, respectively.

Once mosquito data is retrieved to the lab, proceed to mosquito species identification using morphological keys (Annex 1, Annex 4.6).

Table 1. Proposed weekly trapping schedule for Tier 1 sites.

Tier 1 Sites Trap Types Used 1st Trap Visit of The Subsequent Weekly Week Trap Sample Removal • ABWP Airport Minimum requirement: Monday early morning: • Wednesday morning • PAG Commercial • 1 BG-Sentinel w/ or • Remove catch-bag • Remove catch-bag Port w/o BG-Lure, and replace battery, and replace battery, • GMHA • 2 ovitraps w/ hay • Check ovitraps • Check ovitraps, infusion. paddles for eggs and paddles for eggs and condition, replace condition, replace hay infusion. hay infusion. • Friday morning: pick- up traps.** Astumbo Area Minimum requirement: Begin Thursday (morning Every 5-6 days: 1. Middle and 1. 6 ovitraps w/ hay or afternoon): 1. Check ovitraps elementary infusion (3 traps in 1. Check ovitraps paddles for eggs and schools each school). paddles for eggs and condition, replace 2. Residential area 2. 3 ovitraps w/ hay condition, replace hay infusion. infusion; hay infusion. 2. Check ovitraps; larval/pupal 2. Check ovitraps; conduct surveys*. conduct opportunistic opportunistic larval/pupal surveys. larval/pupal surveys. GRMC Hospital Minimum requirement: Begin Thursday (morning Every 5-6 days: • 5 ovitraps w/ hay or afternoon): • Check ovitraps infusion. • Check ovitraps paddles for eggs and paddles for eggs and condition, replace condition, replace hay infusion. hay infusion.

*Larval/pupal surveys are highly recommended for the residential Astumbo area to ensure that larval/pupal surveys yield mosquito data similar to mosquito data yielded by the ovitraps, as the Astumbo area is heavily littered with a variety of water-holding containers (kitchen appliances, tires). Moreover, larval/pupal surveys promote source reduction activities.

**Note that in the ideal scenario, traps should be left out even during the week-end. However, because DEH does not yet have the equipment for a permanent power supply, the traps will have to be packed on Friday morning, and re-installed on the following Monday.

Annex 3.2. Vector Surveillance Protocol in Tier 2 Sites.

Tier 2 sites surveillance should prioritize the following sites:

1. Hotels along Pale San Vitores Road in Tumon (e.g., Hilton Guam Resort and Spa, Governor Josef Flores’ Park, Fiesta Resort Guam, Pacific Island Club Guam, Pacific Star Resort and Spa, Guma Trankilidat Guam Housing, etc.), 2. Chamorro Village and Paseo De Susanna (Hagåtña), and 3. Sheraton Laguna Guam Resort (Tamuning).

Refer to Annex 3.2.i for a complete list of Tier 2 sites. If resources permit, additional Tier 2 sites may be added. Priority should lie on residential areas and populated communal areas such as schools and hotels.

Tier 2 sites vector surveillance should be conducted at minimum on a bi-weekly basis, may be conducted with ovitraps only, supplemented with larval/pupal surveys. In the ideal case, DEH should conduct Tier 2 surveillance at least every 2 weeks. If staff resources do not permit this, DEH should aim to conduct Tier 2 surveillance at least every 3 weeks. Table 1 below outlines a proposed schedule for Tier 2 surveillance.

Refer to the trapping protocols (Annex 4.4 and Annex 4.5) for proper use of ovitraps for egg, and larval/pupal surveillance. Field data must be recorded during every collecting trip. Field data recording templates were written with PIHOA entomologist and DEH staff (Annex 5.1, Annex 5.2). The field data sheets ensure that all necessary information be collected to build important baseline data and to report results to CDC’s MosquitoNET. Field data sheets should be digitized for safekeeping.

Transport of mosquito specimens from ovitraps in the field back to the laboratory should abide to Annex 6.2. Once mosquito data retrieved to the lab, proceed to mosquito species identification using morphological keys (Annex 1, Annex 4.6).

Table 1. Proposed trapping schedule for Tier 2 sites surveillance.

Tier 2 Sites Trap Types Used 1st Trap Visit of the Subsequent Weekly Week Trap Sample Removal • Hotels along Pale Minimum requirement: Conduct larval/pupal N/A. San Vitores Road • Larval/pupal survey once during (Tumon) surveys. week. • Sheraton Laguna Guam Resort Chamorro Village Minimum requirement: Monday: Friday (of the same • 6-10 ovitraps w/ • Set up ovitraps w/ week): hay infusion. hay infusion. • Pick up paddles and traps.

Annex 3.2.i. Additional Tier 2 Sites.

• Pia Marine Hotel and Condominium (Tumon), • Harmon Industrial Park (Harmon), • Two Lovers Point (Dededo), • Perez Acres (Dededo), • Agana Heights Elementary School (Agana Heights), • Skinner’s Plaza (Hagåtña), • Dededo Sports Complex (Dededo), • Dulce Nombre de Maria Cathedral (Hagåtña), • Guam Museum (Hagåtña), • John F. Kennedy High School (Tamuning), • Jose Rios Middle School (Piti), • New Dededo Flea Market (Dededo), • University of Guam Dormitories (Mangilao), • University of Guam Dean’s Circle (Mangilao), • Barrigada Community Center (Barrigada), • Mong Mong Toto Maite (MTM) Community Center (MTM) • Pigo Catholic Cemetery (Anigua), and • Chamorro Cultural Center (Saggan Kotturan Chamoru) (Tumon)

Annex 3.3. Vector Surveillance Protocol in Tier 3 Sites.

Tier 3 sites are concentrated in the south of the island of Guam. As the south is mostly rural and less densely populated than the north of the island, the risk of mosquito-borne disease transmission is lower in the south. Therefore, MSCP should conduct extensive larval/pupal surveys in Agat, Umatac, Merizo, Inarajan, and Yona for one week once to twice a year preferably during rainy season. Surveys should take place during the rainy season, except in the event of abnormal rainfall during the dry season.

The larval/pupal surveys should be sufficiently thorough to determine:

• Productive mosquito egg-laying sites and container types (e.g., bromeliads); • Aedes albopictus hotspots, particularly around communal areas (e.g., markets and churches), and schools; and • The extent of water-holding container litter.

The MSCP field team should prioritize collecting and outreach around human infrastructures.

Once mosquito data retrieved to the lab, proceed to mosquito species identification using morphological keys (Annex 1, Annex 4.6).

Annex 4.1. Adult Mosquito Trapping Protocol: BG-Sentinel 2 (Biogents, GmbH, Regensburg, Germany).

Revision 1

Purpose: To set up BG-Sentinel 2 (BGSts) for adult Aedes mosquito surveillance in designated high-risk areas. This protocol focuses on collecting important vectors of ZIKV, CHIKV, and DENV, such as Ae. aegypti, Ae. albopictus, and other vectors belonging to the subgenus Stegomyia. The BGSt targets blood-meal seeking females, but attract both males and females.

Equipment: (1) Intake funnel, (2) funnel net, (3) catch bag (2x), (4) trap cover, (5) inner cylinder with attached ventilator, (6) trap body, (7) battery cable, (8) 6 - 12-volt battery, (9) power cord with transformer (optional), (10) BG-Lure cartridge (lactic acid) which is effective for 5 months after opening (optional) (11)

CO2 emitter nozzle (optional), (12) octenol lure (optional), and (13) ice or solidCO2/dry ice (optional). NOTE: Use of any type of lure is entirely optional.

Refer to the BG – Sentinel 2 Mosquito Trap Instruction Manual for full assemblage instructions of BGSt (file:///C:/Users/Owner/Desktop/Mosquito%20Lab/Lab%20documents/Mosquito%20Collecting/SOPs%2 0for%20Guam%20Mosquito%20Lab/BG_Sentinel_2_Manual_EN_web.pdf)

Step 0. If the BGSt is new, it should be aired out for at least 2 weeks before initial use, as mosquitoes are repelled by the smell of new BGSts. Before initial use of BGSt, check the following: (1) BG-Lure presents no leakage from pouch as dried out lures lose their potency, (2) fans are functional and are blowing in right direction, (3) elastics are secure, not slack, and not on the verge of breaking. Ensure batteries are fully charged before going to the field. A fully charged battery should last up to 24 to 48 hours. Lures should be sealed in zipper bags and stored at about -18o C (0o F) when not being used to prolong their useful life.

Step 1. Select sampling site, and determine how many BGSts will be deployed based on available BGSts and field staff. Print out field template (Annex 5.1, see bottom of Annex 4.1 for Legend) to start recording outlined data; print out physical map (Google maps print out is sufficient) to mark trap localities.

Step 2. Upon inspection of sampling site, determine which areas might be conducive to mosquito activity, and therefore, potentially ideal to set up BGSt. Within sampling site, favor coverage of a representative sample of all favorable mosquito sites. When searching for an ideal Aedes mosquito site, look for: (a) standing water bodies, (b) artificial water-holding containers, (c) natural water holding containers such as coconut husks, (d) frequency of human activity, (e) protection from wind, and (f) shaded areas.

Step 3. Then, before placing trap, ensure the site selected minimizes exposure to wind, and in shaded areas. Ensure space is left between the BGSt and the tree or whichever element is shading the trap. Do not place a trap within 2 feet of a wall. Also try to maintain the trap visible to patrolling mosquitoes. If rain is a risk, place trap such that BGSt is protected from rain, or consider placing a waterproof tarpaulin about one meter above the trap.

Step 3.a. For measuring treatment efficacy (i.e., before and after trapping for adulticide): BGSts should be placed at any identified highly productive site 1-2 weeks before treatment, and 1-2 weeks post treatment. Trap should not be placed directly upwind of larval habitats, because this would be using the trap as a control measure, rather than a surveillance measure. In this case, BGSt quantity will fluctuate based on number of traps available and field capacity.

Step 4. To place trap:

1. Release carrying handle to allow trap body to pop up; 2. Attach the funnel net over the ring of the intake funnel; 3. Inspect both catch bags: make sure the seams of the catch bag are facing outwards (to facilitate removal of specimens); 4. Attach the catch bag over the funnel net; 5. Insert the intake funnel (with catch bag downwards) into the intake funnel; 6. Rotate funnel clockwise till in locks in place; 7. Remove the label from the BG cartridge and write the date on top of the lure with a permanent marker; 8. Pop out the white disk from the whole in the cover and insert the BG-Lure cartridge; 9. Slide the ventilator cable through the opening on the side of the trap; 10. Lock the trap cover to the body of the trap by pulling the plastic white tabs up and over the 3 slots on the trap cover; 11. Connect the open ends of the ventilator cable with the battery/outlet cable and twist the locking ring tight; 12. Connect the battery cable to a 6 - 12-volt battery or plug outlet cable to power source; and 13. Attach any warning sign the trap body. Refer to Annex 4.4.i for trap label template.

Step 5. Traps should have a minimum of 200 meters of separation from each other. Record in the field template (Annex 5.1) coordinates of trap, trap number (trap numbers should never be changed, each trap should maintain its unique identification number throughout all surveillance activities), and other relevant information as outlined by template.

Step 6. Mark traps on a physical map. Use a colored masking tape to mark trap locations (tie to a nearby tree branch or pole).

Step 7. Traps should be surveyed every 24 - 48 hrs. Any more than this and specimens may die and dry out, or battery power may be too weak to retain captured mosquitoes in the catch-bag.

Step 8. When removing and/or replacing the catch bag, the fan must be maintained to ensure no mosquitoes escape from the catch bag:

1. Bring with a fully charged 6 - 12-volt battery, a cooler with ice/dry ice, the second catch bag; 2. Lock the shutter of the intake funnel by pressing down on the locking tab; 3. Remove the intake funnel by rotating it counter-clockwise and pulling up; 4. While holding the funnel over the suction of the fan, carefully slide the catch bag off the intake funnel while simultaneously pulling the draw string tight; 5. Handle the catch bag carefully to not damage and crush mosquitoes; 6. Place the catch bag in a Tupperware sitting in a cooler on ice (Refer to Annex 6.1 for transportation of specimens protocol); 7. Replace the catch bag on the intake funnel and remember to release the locking tab; and 8. Replace the used battery with a fully charged battery. Notes: As BGSts are an essential trapping method for surveillance of adult Aedes, it is advisable to keep in storage extra parts in case a replacement is necessary: gauze coverings, catch pipes, fans, wire, battery

clips. Recommendation: Using a permanent power supply is advised to help alleviate trap visit frequency and to ensure sustained power supply to the fan.

Legend of Annex 5.1.

Legend Locality: write village and island name, add nearest street address (ALWAYS – MosquitoNET requires) L/NL: Littered or Not littered, i.e. presence of artificial containers or absence of artificial containers. Habitat: beach, forest, open (sparse tree cover); Soil type: muddy, sandy, mesic (dry); Sun exposure: shady vs sunny. R/C: Residential or Commercial. Egg count: count eggs when surveying ovitraps.

Annex 4.2. Adult Mosquito Trapping Protocol: CDC Miniature Light Trap.

Revision 1

Purpose: Primarily to, but not exclusive of-, collect non-Aedes female mosquitoes attracted to the light enhanced with CO2 attractant (dry ice), such as Culex spp., which are known vectors of lymphatic filariasis, and Anopheles spp., which are known vectors of Plasmodium parasite (i.e., malaria). Relevant to mosquito surveillance, this trap can be used for monitoring and assessing the effectiveness of vector control activities, and for determining relative abundance of mosquitoes.

*Note: CDC miniature light traps are recorded to collected few Cx. quinquefasciatus; however, results vary considerably across geographic regions, and thus, Guam should monitor trap counts until enough data is recorded to determine usefulness of CDC mini light trap for capturing Cx. quinquefasciatus.

Equipment: Designed to be portable, weighs 1.75 pounds, and functions with: (1) four D cell (1.5 volt) batteries OR, one 6 -12-volt motorcycle battery (either options supply a single full night of operation, and should be expected to operate for 15 - 25 nights before batteries wear out and need to be changed), (2) double-ring large fine-mesh catch bag, (3) collection body, and (4) collection container. Note: Trap may be combined with CO2 as supplementary attractant (dry ice).

Step 0. Test the trap to make sure the light and fan are both working by connecting the black and red cables to their 6-volt connections on the battery. Disconnect battery after testing.

Set up trap by attaching collection container or net bag to the body of a trap.

Step 1. Select sampling site according to the Guam Vector Management Strategic Plan and determine how many CDC mini light traps will be deployed based on available traps and field staff. Print out template (Annex 5.1) to start recording outlined data; print out physical map (Google maps print out is sufficient) to mark trap localities.

Step 2. Upon inspection of sampling site, determine which areas might be conducive to mosquito activity, and therefore, potentially ideal to set up CDC mini light trap. Within sampling site, favor covering a representative sample of all favorable mosquito sites. When searching for an ideal Culex mosquito site, look for: (a) oviposition sites (fresh, stagnant water, and although this is not mandatory, but will increase the odds of trapping females which have just had at least one blood meal and are more likely inoculated with a pathogen), (b) shelter from wind (tall grasses, weeds), (c) any artificial and natural containers. Note that Anopheles prefer: (a) clean, unpolluted water, (b) fresh- or salt-water marshes and other marsh-type habitats, grassy ditches, edges of streams and rivers, and small, temporary rain pools, (c) habitat with vegetation, or lack of, depending on species, and (d) open, sun-lit pools, or shaded breeding sites in forested areas.

Step 2a. If traps are being used with the light, ensure traps are not placed near other light sources.

Step 2b. If there are no hanging devices available in the desired sampling location, improvise a hanging device such as a tripod.

Step 2c. Reconnect the battery after the trap is installed.

Step 3. Traps should be in position and operational from just before dark, until just after daylight. Place trap at least 6 feet above ground level from a post or tree, 30 feet or more from buildings, in open areas

close to trees and shrubs. Traps should be placed at least 100 feet apart from each other. Trap location coordinates and other relevant field data information should be recorded following the provided template in the Guam Vector Management Strategic Plan. Finally, as for BGSts, each CDC Mini light trap should have its unique identification number.

Step 4. Mark traps on a physical map.

Step 5. Traps should be surveyed every 24 hours, and used regularly (1 - 7 nights per week). Batteries will usually last through 1 - 2 nights before they need to be replaced or recharged.

Step 6. To remove the catch bag:

1. Tap the sides of the container to ensure specimens fall to the bottom, 2. Tie the top of the catch bag (i.e., nearest to the light sources), 3. Remove the collection container from the trap, and 4. If the specimens will be killed, immediately place the collection container in a cooler with dry ice for at least 30 minutes. If ice is used, leave for at least an hour to two. Refer to Annex 6.1 for transport of specimens back to lab.

Annex 4.3. Adult Mosquito Trapping Protocol: CDC Gravid Trap.

Revision 1

Purpose: Particularly effective for collecting virus-positive mosquitoes, specifically attracts gravid females. Thus, the purpose of this trap is to monitor viral activity. This trap favors the collection of Culex spp., but also some Aedes, and to a lesser extent, Anopheles spp.

Equipment: A plastic washbasin containing liquid oviposition attractant (i.e., ‘Mosquito Soup’); with a device on top which pulls air through a collection chamber (trap tube and metal support), thus preserving the mosquito specimen. Lured by the stagnant water, a female mosquito will try depositing her eggs on the surface of the water to lay her eggs. The trap runs on a 6 volt or D cell battery to work the fan.

Step 0. Ensure batteries are charged and suitable for use.

Step 1. Prepare Mosquito Soup. The standard mosquito soup recipe calls for 1 pound of hay, 1 ounce of albumen (malted milk powder), and 1 ounce of brewer’s yeast, for 30 gallons of water, and incubated for 5 days. However, entomological field work in the Pacific islands indicate that using only grass clippings (or perhaps palm fronds) is sufficient. The recommended mosquito soup recipe is as follows: (1) 50 grams per liter of water (but for Culex, 80 grams), (2) Let sit for 5 days, mix well, and (3) Filter water (with cheese cloth).

Step 2. If not already done, puncture holes at the top of the washtub, near the rim, to avoid overflow in the event of rain.

Step 3. Print out field data sheet template (Annex 5.1) to start recording outlined data; print out physical map (Google maps print out is sufficient) to mark trap localities.

Step 4. Upon inspection of sampling site, determine which areas might be conducive to mosquito activity. Within sampling site, favor coverage of a representative sample of all favorable mosquito sites. Look out for: (a) sheltered areas/protected from the sun, (b) urban and vegetated areas, (c) unkempt areas, and (d) areas where birds congregate. NOTE: keep far away enough from other breeding sites to avoid breeding site competition.

Step 5. Set up trap carefully on the ground following these sequential steps: (1) set washtub and secure trap and capture system over the tub; tube must be about ½ an inch above the water surface (2) set battery, (3) cautiously pour mosquito soup in washtub to fill only a few inches of the washtub (no need to overfill), (4) plug in battery, and (5) optional: place a 13-gallon plastic bag punctured with breathing holes over the collecting to protect mosquitoes from rain

Step 6. As traps are low on the ground, they might be hard to locate. Thus, use bright colored flagging tape to tie to tree branch to indicate presence of a trap.

Step 7. Record trap locality coordinates and all relevant field data as outlined by the template provided in the Guam Mosquito Surveillance Plan. Ensure all Gravid Mosquito Traps have and preserve their unique identification number.

Step 8. Mark traps on a physical map.

Step 9. Let trap run for a minimum of 1 night, and a few feet apart from each other. Traps should be surveyed early the following morning to ensure maximum survival of the mosquitoes.

Step 10. To remove specimens from trap:

1. Ensure fan is still running; 2. Slide the net up the trap while fan running; 3. Close off net with hands to ensure no mosquitoes fly out; 4. Once free of the trap tube, tie off the string closing the mouth of the trap; and 5. Place specimens in dry ice coolers by collapsing net. *Note: (1) The trap may have a suspicious appearance and may be worrying to the public. Thus, consulting with the owners/responsible person regarding the sampling site is recommended. In addition, affix a sign describing the trap and its intended purpose. The sign should include DEH’s phone number the public can call with questions/concerns. (2) The hay infusion may be very heavy to carry.

Annex 4.4. Mosquito Egg Trapping Protocol: Ovitraps and Hay Infusion.

Revision 1

Purpose: To outline how to (1) make hay infusion, (2) make ovitraps, and (3) place ovitraps for surveillance.

Equipment: Hay infusion: hay, blender, 1-gallon water bottles for storages; Ovitraps: plastic water bottles, black duct tape, scissors, paper, clear duct tape, tongue depressors, brown paper towel, and rubber bands.

Step 0. (I) Make hay infusion: (1) Blend a cup of hay in a blender till very fine; (2) Place hay infusion in a Tupperware; (3) Fill a 1-gallon water bottle with clear water. Do not overfill.; (4) Pour 1 tablespoon of blended hay into the water bottle; (5) Close bottle and shake; and (6) Let infuse at least 24 hours.

(II) Make ovitraps/ovicups: (1) Cut top of plastic water bottle (size does not matter) and dispose of top; (2) Remove bottle label; (3) wrap bottle in black duct tape; (4) pierce four holes the size of a pea at the rim of each ovicup to prevent rain overflow; (5) Place the label on the bottle; and (6) use clear tape over the label to protect it from rain. Refer to Annex 4.4.i for trap label template.

(III) Make ovitraps paddles: (1) Wrap a single sheet of brown paper towel around a tongue depressor: leave about an inch at both ends of the tongue depressor exposed; (2) Use rubber bands to secure the paper towel onto the tongue depressor on both ends (Figure 1); and (3) label each paddle with a letter code, such as A, B, C, etc., with a water-proof permanent marker.

Figure 1. Ovitrap paddle.

Step 1. For the field, bring a clip board with a field data recording sheet (Annex 5.1), at least one 1-gallon bottle with hay infusion, the desired number of ovitraps (and some extras in case some are damaged), the desired number of paddles (use 1 or 2 ovipaddles per ovitrap), extra rubber bands, pens, and brightly colored masking tape.

Step 2. In the field, place ovitraps in shaded areas, preferably protected from the rain. Ovicups may be placed indoors and/or outdoors. Placing ovitraps indoors is especially effective for collecting eggs of important Aedes vector species, such as Ae. aegypti. Ensure that traps are never entirely covered by foliage of shelter; leave at least 1 foot between bush or shelter above the ovitrap. Always place ovitraps on the ground.

Tip: If possible, it is suggested to speak to personnel of location being sampled to advise maintenance staff not to throw out ovitraps.

Step 3. When pouring hay infusion in ovitraps, only fill up to about 1/3 - 2/3 of the ovitrap. Ensure ovitrap is stable.

Step 4. Ovitraps may be left outside from 5 to 6 days without being checked. Left out beyond 6 days, the ovitraps will be breeding mosquitoes. Therefore, check the ovitraps and replace the paddles every 5 to 6 days.

Step 5. Ovitraps surveying. Replace the hay infusion if the water appears dirty. Replace the paddles if the paper is dirty or if eggs are visible. Replace rubber bands as necessary.

Step 6. Transport paddles back to lab. Refer to Annex 6.2 for procedure outline.

Annex 4.4.i. Trap Sign/Label Template. Property of Guam Department of Public Health & Social Services (DPHSS) MOSQUITO TRAP - PLEASE DO NOT TOUCH Questions, call Guam Division of Environmental Health: 735 – 7221 This mosquito trap does not pose any health threat to humans and animals

Annex 4.5. Mosquito Larval/Pupal Surveillance: Conducting a Larval/Pupal Survey.

Revision 1

Purpose: To outline the key points of conducting a larval/pupal survey for surveillance of important vector mosquito species for humans, and to outline how to process collected samples in the lab (i.e., vial content transfer to rearing chamber).

Equipment: Field: plastic medium-sized vials or Whirl-packs® (Nasco), dippers, turkey basters, transfer pipettes, plastic trays, water bottle(s) with clear tap water, backpack, labels for vials or for Whirl-packs®, clipboard, pen, and larval/pupal survey forms (Annex 5.2). Lab: 1-gallon dechlorinated water (tap water may be allowed to sit to allow chlorine to dissipate), transfer pipettes, rearing chambers, environmental chamber, larval food, cotton balls, sugar-water solution (10% sucrose), labels or label tape.

Recommended field equipment: DEET (mosquito repellent); raincoat; sunglasses; sunhat; sunblock; lightly-colored, long-sleeved shirt/pants, and hiking boots.

Step 0.

• When going out in the field to conduct a larval/pupal survey, pack a backpack with the items required. It is preferable to go with at least one other person to assist with completing the larval/pupal field sheet and placing labels on vials. • Larval/pupal surveys are to be conducted as outlined in Annex 3.1, 3.2, 3.3, 7, 8, and 9. • Prepare the larval/pupal field datasheet to indicate the collection code, site of sampling, etc.

Step 1. Identify artificial and water-holding containers. Water-holding containers include: tires, buckets, saucers, ponding basin, culvert systems, ground puddles, rain catchments, flower pots, pans, pet drinking bowls, birdbaths, shallow pools, bromeliads, coconut shells, palm fronds, coconut husks, bamboo, rock pools, tree holes, roof gutters, etc. Prioritize water-holding containers that are situated near human residences, hotels, hospitals, and schools. Check for presence/absence of water.

Step 2. If water is present, examine it to identify if mosquito larvae and/or pupae are present/absent.

• If the water-holding container is large, e.g., large ponding basin, wetland, etc., use dipper to scoop water into the plastic tray. Check for larvae/pupae. • If the water is too murky to determine larvae/pupae presence/absence, stir the murky water and use a dipper or a turkey baster to collect a sample. Transfer the water into a plastic tray and mix this volume with clear water. Rock the mixture back and forth, allow particles to settle, and examine for larvae/pupae presence.

Step 3. If larvae/pupae are present in the container, use a turkey baster to collect a representative sample. Transfer the sample into a vial or Whirl-pack®:

• Agitate water by “mixing” the water with the turkey baster. • Take samples from different water levels of the water column, as different larval species either feed below or at the surface of the water.

Step 4. Securely close the vial. Place a label on the vial. The label should indicate the place, date, and container-type of sample collected. Samples from the same site and same container-type may be pooled. DO NOT USE THE SAME VIAL FOR DIFFERENT CONTAINER-TYPES.

Step 5. Use the larval/pupal survey datasheet (Annex 5.2) to tally the container-types that contain water, are surveyed, have larvae or pupae present, and if relevant, the whether there are more or fewer than 12 pupae. If there are fewer than 12 pupae, count the exact number of individual pupae. If there are more than 12 pupae, indicate this observation by marking “>12”. This is important because the presence of pupae indicates that the container type sampled has environmental conditions favorable to larval survival.

Step 6. Place the vial in a backpack. Ensure that the samples are kept safe from heat or turbulence. Continue with survey.

Step 7. The more water-holding containers are sampled, the better. There is no “right or wrong” number of containers to sample. As a guide, if sampling containers in a single 2-lane street and if time-constrained, aim to sample containers at every other house. At a hotel, aim to sample all containers, but if not feasible, sample all containers surrounding main entrances and exits, dining areas, and other recreational areas where people congregate.

Step 8. Back at the lab. When returning from the field with the samples, immediately place the vial contents in rearing chambers. For a single sampling site, place the contents of multiple vials of the same container-type in a shared rearing chamber. However, if samples were collected from different sampling sites, do not share sample contents from different sites in a single rearing chamber.

Step 9. Placing vial content in a rearing chamber.

1. Ensure the rearing chamber is properly cleaned before use and the funnel is in the correct position (Figure 1);

Figure 1. Correct placement of rearing chamber components.

b. c.

2. Open the vial, and carefully pour the contents into the bottom chamber of the rearing chamber; 3. If needed, add in some dechlorinated tap water from the 1-gallon water bottle; 4. With a small transfer pipette, remove non-mosquito insect species, such as nematodes, midges, water and beetles; 5. If large numbers of copepods (small crustaceans) are observed (Figure 2), use a transfer pipette to transfer the mosquito larvae to another rearing chamber, as copepods may be aggressive towards mosquito larvae;

Figure 2. Copepods.

6. If it is observed that a larva attacks other larvae, it may be a carnivorous, and should be placed in a separate rearing chamber. Use a transfer pipette for this procedure; 7. If the water does not have enough organic matter for the larvae to feed on, add a minimal amount fish food (small pinch), or any other type of food sources that are appropriate (e.g., ground up dog food, liver powder, etc). o It is important that the food source is not contaminated with mold or too fatty for the larvae. DO NOT OVERFEED LARVAE; 8. If possible, transfer the label from the vial to the rearing chamber. Otherwise, transfer the information to a new label and adhere to the rearing chamber; 9. Close and hand-tighten the rearing chamber. Do not overtighten; 10. Place the rearing chamber into environmental chamber set at 27.5 - 28o C (81.5 – 82.4 o F) with a day/night cycle set at 6:00 a.m. to 6:00 p.m.; 11. Repeat steps 1-10 as necessary; 12. Place a cotton ball soaked in a 10% sugar-water solution on the top of the rearing chamber to attract the emerged adult mosquitoes to the top chamber of the rearing chamber and feed them. 13. Check on larvae daily during the work week. Feed larvae as needed. 14. Use a log sheet to record the date the larvae were last fed, temperature of environmental chamber, and person who conducted last feeding. Note that in artificial environmental conditions, mosquito larvae/pupae may take longer than “normal” to complete life cycle development. Therefore, monitoring the conditions under which mosquitoes are reared to adults is important to determine optimal conditions for mosquito rearing.

Annex 4.6. Adult Mosquito Species Identification Guidelines.

Revision 1

Purpose: To outline the step-by-step process to identifying unpinned/pinned adult mosquito specimens to (1) genus, and (2) to species. This protocol is based on the guidance provided by Dr. Harry Savage (US CDC, Fort Collins, CO).

Equipment: Prepare the work station. For adult mosquito identification, a dissecting microscope with 40x maginification is sufficient (a compound microscope is only necessary to examine larval/pupal skins). Also needed are label paper, scissors, pins, mounting block, and the appropriate mosquito species identification keys. Keep copies of mosquito anatomy diagrams and abbreviations handy for reference. Keep these diagrams at the dedicated taxonomic workbench. Also ensure all the taxonomic identification keys listed in Table 2 of this protocol are available for reference.

Table 1. Genera and subgenera present on Guam.

Genus and subgenus Abbreviations Anopheles (Anopheles) An. (Ano). Anopheles (Cellia) An. (Cel). Aedomyia (Aedomyia) Ad. (Ady). Aedes (Aedimorphus) Ae. (Adm). Aedes (Stegomyia) Ae. (Stg). Armigeres (Armigeres) Ar. (Arm). Culex (Culex) Cx. (Cux). Lutzia (Metalutzia) Lt. (Mlt). Mansonia (Mansonioides) Ma. (Mnd). Wyeomyia (Wyeomyia) Wy. (Wyo).

Table 2. Mosquito classification of recorded mosquitoes on Guam (Order: Diptera; Family: Culicidae).

Subfamily Tribe Genus Subgenus Group Species Anophelinae Null Anopheles Anopheles barbirostris barbirostris Anophelinae Null Anopheles Cellia Pyretophorus • indefinitus series • subpictus • vagus Anophelinae Null Anopheles Cellia ludlowae litoralis Culicinae Aedomyiini Aedomyia Aedomyia Null catasticta Culicinae Aedini Aedes Aedimorphus oakleyi Culicinae Aedini Aedes Aedimorphus vexans vexans nocturnus Culicinae Aedini Aedes Stegomyia aegypti aegypti Culicinae Aedini Aedes Stegomyia scutellaris • albopictus • guamensis Culicinae Aedini Aedes Stegomyia pandani • pandani • rotanus • saipanensis Culicinae Aedini Armigeres Armigeres subalbatus subalbatus Culicinae Culicini Culex Culex pipiens • quinquefasciatus

• fuscocephalus Culicinae Culicini Culex Culex sitiens • sitiens • annulirostris marianae • tritaeniorhynchus • litoralis Culicinae Culicini Lutzia Metalutzia fuscana Culicinae Mansoniini Mansonia Mansonioides uniformis Culicinae Sabethini Wyeomyia Wyeomyia mitchellii

Table 3. Important mosquito identification taxonomic keys*.

Authors and Year Title Guillaumot, L. (2015). Key to the mosquitoes of Guam. Bohart, R. M. (1956). Insects of Micronesia, Diptera: Culicidae. Yamaguti & LaCasse (1950). Mosquito fauna of Guam. Rattanarithikul, et al. (2005). Illustrated keys to the mosquitoes of Thailand. 1. Background; geographic distribution, list of genera, subgenera, and species; and a key to genera. Rattanarithikul, et al. (2005). Illustrated keys […] II. Culex and Lutzia. Rattanarithikul, et al. (2006). Illustrated keys […] III. Aedomyia and Mansonia. Rattanarithikul, et al. (2006). Illustrated keys […] IV. Anopheles. Rattanarithikul, et al. (2010). Illustrated keys […] VI. Tripe Aedini. Tanaka, et al. (1979). A revision of the mosquitoes of Japan. Sirivanakan, S. (1976). A revision of the subgenus Culex in the Oriental region (Diptera: Culicidae). Cagampang-Ramos & Darsie (1970) Illustrated key to the Anopheles of Philippine Islands. Darsie & Ward (1981). Identification and geographical distribution of the mosquitoes of North America, north of Mexico. Clark-Gill & Darsie (1983). The mosquitoes of Guatemala, their identification, distribution, and bionomics, with keys to adult females and larvae. *Copies of these keys have been provided by Dr. Harry Savage, CDC to the DEH staff on a flash-drive. Additional keys are also included in the distributed flash-drive.

Step 1. Separate the mosquitoes from the non-mosquitoes. Dump the non-mosquitoes into the rubbish bin. Reminder: mosquitoes have an elongated and pointed proboscis (mouthpart), 2 wings and a pair of halters, abdomen not curled upwards, and can be small to medium-large (smaller than crane flies, which have much lankier and longer legs) (Figure 1).

Figure 1. Crane fly (a) versus mosquito (b).

a. b.

Step 2. Taxonomic keys are written for the identification of female mosquitoes. Males may be sorted to genus using the “Bohart 1956” key. Focus should be placed in identifying female mosquitoes over male mosquitoes. Separate the male mosquitoes from the female mosquitoes. Male mosquitoes have plumose antennae, which give their antennae a feather-like appearance (Figure 2). Set males aside in a petri dish, ensuring they are properly labelled. Store males in freezer for identification if necessary.

Figure 2. Male versus female mosquito antennae.

Step 3. Identify female specimens to genus. Use the key to genus by Guillaumot (2015) if necessary (see Annex 1 for link), Bohart (1956), and Rattanarithikul et al (2005).

Place each genus identified in separate petri dishes to make identification process easier. Work on one genus at a time.

• If necessary, pin/mount select specimens (or at least one specimen) for the ease of manipulating a mounted specimen rather than continuously rubbing unpinned/unmounted specimens on the petri dish while manipulating the specimens to examine the required characteristics.

Step 4. Begin genera identification with Bohart (1956). The most widely collected genera on Guam are Aedes and Culex.

• Determine whether the specimen(s) is/are either of these genera using the Bohart (1956) keys. If it is not either, see Step 5. • If the specimens examined belong to Aedes: 1. Identify to species using Bohart (1956). 2. If this is the first time the species is observed by the identifier, always double-check species identification by using other keys, as indicated in Table 3.

3. If necessary, send specimens to Walter Reed Biosystematics Unit for species identification confirmation. • If the specimens examined belong to Culex: 1. Determine whether species belong to the pipiens group or to the sitiens group: use the Sirivanakan (1976) key to species group. 2. If specimens belong to sitiens group, use Bohart (1956) to determine species. 3. If specimens belong to pipiens group, continue to species identification with Sirivanakan (1976). Double-check the findings with Rattanarithikul et al (2005). 4. If unsure of the species the specimens are, simply indicate the genus, subgenus, and species group (e.g. Cx. (Cux.) pipiens gp.). This is already a good identification, as certain Culex species are very difficult to identify to species. 5. If necessary or if it is a novel species to Guam, send the specimen(s) to Walter Reed Biosystematics Unit for species identification confirmation.

Step 5. If neither Aedes nor Culex, use Bohart (1956) to determine if the mosquito is one of the following: Anopheles, Aedomyia, Armigeres, Mansonia, or Lutzia. If the mosquito does not key out, compare the specimen to Wyeomyia in the reference collection.

Follow the recommendations below:

• If Anopheles: 1. Begin by using Rattanarithikul et al (2006) key for Anopheles to determine whether Anopheles specimens belong to subgenus Anopheles or subgenus Cellia; 2. If the Anopheles specimens belong to subgenus Anopheles, continue to species identification with Rattanarithikul et al (2006) key for Anopheles; 3. If the Anopheles specimens belong to the subgenus Cellia, continue to species identification with Cagampang-Ramos & Darsie (1970); 4. If unsure of the species, indicate the genus, subgenus, and if applicable the species group; and 5. If necessary, send specimens to Walter Reed Biosystematics Unit for species identification confirmation. • If Aedomyia: 1. Use Bohart (1956). 2. The only Aedomyia species recorded on Guam is easily recognizable: ▪ Tufts on hind femur; ▪ Wings are with yellow and white scales. ▪ The antennae are shorter. ▪ Absence of pre- and post-spiracular setae. ▪ Larvae are distinctively green. • If Armigeres: 1. Will not key in Bohart (1956). 2. Use Tanaka et al (1979). 3. Double-check with Rattanarithikul et al (2010).

4. Tip: appearance similar to Aedes, but no stripes on female’s thorax, and proboscis curved downwards. • If Mansonia: 1. Recognizable due to their large size and heavy scaling. 2. Use Rattanarithikul et al (2006) III. 3. Use illustrations in Tanaka et al (1979) for visual aid. • If Lutzia: 1. Reminder: many older keys will consider this species as a member of the genus Culex. 2. Use Bohart (1956). 3. Use Guillaumot (2015). • If Wyeomyia: 1. This species will look very different from the others. 2. Compare the specimens to those in the reference collection. 3. Use Bohart (1956) to ensure it does not key to any other species. 4. If Wyeomyia mitchellii is suspected, refer to: (1) the online interactive key of the WRBU (see Annex 1 for link), then confirm with (2) Darsie and Ward (1981), and (3) Clark-Gil and Ward (1983).

Annex 4.7. Locality and Det Labels Protocol and Templates.

Revision 1

Purpose: To outline how to produce Locality and Det labels for pinned/mounted mosquito specimens.

Equipment: Good quality cardstock (36-40 pounds minimum, 60 pounds at best); label pens; scissors; and a printer.

Step 1. Make Locality Label (see Annex 4.7.i for a template).

1. Open Microsoft Word. 2. Open a blank and standard Word sheet. Keep in Portrait format. 3. Click on ‘Layout’ tab at top of document. 4. Click on ‘Columns’ → Scroll down drop-down menu and select ‘More Columns’. 5. In ‘Number of Columns’, type in 9 (for 9 columns). 6. In ‘Col #’, for width, type in 0.79”, and for spacing, type in 0.1 for Column #1 only. It will automatically set this format for the other 8 columns. 7. Make sure the ‘Equal Column Width’ box is check-marked. 8. Click OK. 9. Next, in the same ‘Layout’ tab of the document, click on ‘Margins’. 10. Scroll down the drop-down ‘Margins’ menu and select ‘Custom Margins’. 11. Under the heading ‘margins’ of the pop-up window, type in 0.3” for ‘Top’, ‘Left’, ‘Bottom’, ‘Right’. 12. Click OK. 13. Now set the font to Arial. 14. Now set the font size to 3.5 pt. Manually type this in; it is not a selection in the font size drop- down menu. 15. Type in the locality label information. The following information must be included:

COUNTRY: lesser political unit: exact locality (latitude and longitude) date(s), collector(s), collecting method/trap type, habitat field code or unique identifier

Below are some examples:

Label etiquette:

• Labels will generally have 4-5 rows of data, depending on how much information included. More importantly, remain consistent in the label format. • The month of collection date is always in roman numerals (lower case), e.g., August is written as ‘viii’. • The font should always be sans serif.

Step 2. Type in the label information.

1. Zoom in till comfortable in seeing what is being typed. 2. Begin in column 1. Type in the complete label information for label 1. 3. In the same column 1, type in the complete label information for label 2. 4. Select label 1 and label 2. 5. Right click and select ‘Copy’, 6. Directly below label 2, right click, and select ‘Paste’. 7. Repeat till the bottom of column 1 is reached. 8. Select the entire column 1. 9. Right click and select ‘Copy’, 10. Place the cursor at the top of column 2. 11. Right click and select ‘Paste’. 12. Repeat this for column 3, 4, 5, 6, 7, 8, 9. 13. Ensure the label is never separated into different columns. 14. If more than 9 columns of this particular label is needed, repeat as necessary on the next page.

Step 3. Print labels. Printed labels should be no larger than 17 mm long by 6 mm wide.

Step 4. Cut-out the printed locality labels.

1. Printed labels should be cut as closely as possible to the printed text so that extra white space is minimized. However, care must also be taken to avoid cutting pieces off lines of text. Although guillotine-type paper cutters are fast and efficient, they can produce rough or bent edges on labels and increase the chance of cutting off pieces of text or leaving wide margins around the edge of the label. 2. Cut-out each column. 3. Cut-out as many labels as possible. 4. Place in petri dishes for future use. See Figure 1.

Figure 1. Cutting out locality labels.

Step 5. Prepare the Det labels (See Annex 4.7.ii for Det Label template). Use the same paper and etiquette as for Locality labels.

1. Use Template provided in Annex 4.7.ii. 2. Keep format as is. 3. If necessary, copy-paste the Annex 4.7.ii onto a separate word document, set the Margins as Top: 0.25”, Bottom: “0.25”, Left: 0.38”, Right: 0.5”. Set the Header and Footer at 0.2”. 4. In the first square, replace template name with the determiner’s name and current year (i.e., year of specimen identification). 5. Copy-paste the edited square to the entire document, to have the determiner’s name and year in all the boxes.

Step 6. Print labels. Utilize white or cream cardstock or similar gaged paper for printing the labels.

Step 7. Cut-out labels.

1. Cut on the outer edge of the black framing of the label. Cut-out more than enough. 2. Store in petri dish. 3. For det labels, the name of the species is hand-written with label pens. Ensure the archival ballpoint is small, such as 01, 02, 03.

USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC USA: GU: Astumbo Elementary School along fence, (13.554830, 144.947891), BG-Sentinel w/ BG-Lure, viii.17.2017, Guam DEH & CDC

Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017 Det: E. Vajda, 2017

Annex 4.8. Pinning/Mounting of Adult Mosquito Specimens.

Revision 1

Purpose: To outline how to properly pin adult mosquito specimens and include labels.

Equipment: Pinning block; No.3 (or No. 1, No.2) pins; point paper; point hole puncher; Det labels; Locality labels; foam block (or cork pad if no foam block); white Elmer’s glue; white paper; soft forceps; petri dishes to use as containers; scissors; and a good light source.

Figure 1. Equipment for pinning/mounting of adult mosquito specimens.

Step 1. Punch out pointes. With the point hole puncher, punch out as many points as possible and place in a labelled petri dish as in Figure 1.

Step 2. Prepare the point-making work station, as in Figure 2.

Figure 2. Point-making work station: pinning block, No. 3 pins, cork pad with points.

Step 3. Make points.

1. Ensure points are placed on cork block. 2. Bring the pointy part of the pin towards the blunt end of the point. 3. Lightly prick, then push pin through the blunt end of the point. See Figure 3.

Figure 3. Prick, and push pin through paper pointe.

a. b.

4. Place pin with point on highest step of pinning block. Lightly push pin through the hole of the step, as in Figure 4. Do not force pin.

Figure 4. Finishing pin. a. b.

Step 4. Place finished point onto the corck pad. Repeat steps 1 through 3 till there are enough pins for a number of specimens.

Step 5. Pin the specimens. Place mosquito on a white piece of paper (or even a kim whipe will work). The specimen must be on its side, with legs pointing down towards the pinner, and head towards the pinner’s left. See Figure 5.

Figure 5. Place mosquito specimen on white paper.

Tip of abdomen Head

Step 6. Place a drop of glue on the side of the paper with the soft forceps (Figure 6).

Figure 6. Place drop of glue.

Step 7. Glue specimen.

1. Take the pin by pinching between the fingers slightly below the paper pointe. 2. Lightly dip the point in the drop of glue. 3. Then lightly place the point with glue on the side of the thorax of the mosquito. 4. Hold pin in place for a few seconds to allow the glue to dry onto specimen. 5. Place pinned mosquito on foam block. See Figure 7.

Figure 7. Glueing the specimen.

a. b.

Step 8. Once all the specimens have been, they are ready for (1) identification, and/or (2) if already identified, for labelling (i.e., add labels to pin).

1. Organize the pinned specimens to the match the appropriate locality label for that specimen. On the same cork block, group together all the pinned specimens that will have the same locality label. 2. The labels are ready to be pinned to the specimen.

Step 9. Pin Locality labels.

1. Place the locality label on the cork pad such that the left edge of the label faces down towards the pinner. See Figure 8.

Figure 8. Place locality label with left edge towards the pinner.

2. Then prick the label with the point about ¼ of the label inwards, and slightly off-center. 3. Lightly push the pin through. 4. Bring the pin to pinning block and place it on lowest step. 5. Push the pin through. Do not force. See Figure 9.

Figure 9. Pinning locality label. c.

Step 10. Pinning the Det label.

1. Write the species name on the Det label. Include whether the mosquito is male or female. 2. Each Det label corresponds to a single species. 3. If the Det label only applies to ONE specimen, place the label as in Figure 10.

Figure 10. Place Det label for a single specimen.

4. If the Det label applies for multiple specimens that placed in the drawer/cabinet, place the label as in Figure 11.

Figure 11. Using one Det label for multiple specimens of the same species.

IMPORTANT NOTES:

• Ensure the specimens are evenly spaced between each other such that they do not touch or bump into each other. • Place EPA-approved moth balls in drawer to deter insects (i.e., museum pest species) from infiltrating drawer and eating specimens. • Regularly check drawers for evidence of nibbled specimens. Observe if there are missing body parts, and dark “crumbs” which are visible on the white foam pads of the drawers. o If there is evidence of nibbling on specimens, place all drawers in the freezer for at least 48 hours. This will kill off the museum pest species. o Remove crumbs by dabbing drawer foam pads with one-sided tape. • Regularly check for unglued specimens. This may occur over time. Simply re-glue fallen specimen.

Site Name Site Code Tick Box Annex 5.1. BG-Sentinels, Ovitraps, Gravid Traps, Field Data Recording Template. Port Authority of Guam (PAG) 000001

Form Number: GUAE-17-___ (insert form number here) A.B. Won Pat International Airport 000002

Name of Surveyors: ______Astumbo Elementary School 000003 Guam Memorial Hospital 000004 Signature: ______Guam Regional Medical City 000005 This section is reserved for person entering data only: Date Loaded to CalSurv: ______By: ______CalSurv Collection ID: ______Other: *

Coordinates Trap Type Trap # Lure Date & Time Date & Time L/NL Habitat/Soil Type/Sun Exposure R/C Egg trap set up trap surveyed /Adult count

54 Signature: ______Page ___ of ___

Page ___ of ___ IDENTIFICATION COUNT DATA # CONTAINER TYPE DET. DATE GENUS SPECIES SUBSPECIES FEMAL MALE Entry E ♀ ♂ (Tracker)

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Annex 5.2. Larval/Pupal Surveys Template.

Form Number: GUL-17-___(insert form number) Recommended tools to bring for surveillance/source reduction: Insect repellent (for personal use), larvicide tablets (Bti), knife (for putting holes in containers), flashlight (for looking into dark pools of water).

Date & Time Surveyed: ______Names of Surveyors:______

Site Code Tick Box: Site Name Site Code Tick Box

Port Authority of Guam (PAG) 000001

A.B. Won Pat International Airport 000002

Astumbo Elementary School 000003

Guam Memorial Hospital 000004

Guam Regional Medical City 000005

Other: *

*Person entering data will determine the site code if site is “Other”.

This section is reserved for person entering data only.

Date Loaded to CalSurv: ______By: ______CalSurv Collection ID: ______

DATA SUMMARY: Total containers with water = ______

Total containers with mosquito larvae/pupae = ______

Container name (ex. tire 1) Larvae present # pupae (Y/N)

Comments (ex. Describe if water storage containers are covered and/or maintained, Does the household need help removing sources? Is there a piggery? etc.):

______

PRESENCE OF WATER-HOLDING CONTAINERS (mark with tallies):

# of pupae/ piupa BASIC COLLECTION # of containers # of containers # of containers (if >12, stop CONTAINER TYPES* with water with larvae with pupae counting and write >12) Tires

Plastic containers, buckets

Bromelliads

Flower pots and saucers

Drums (liquid storage container)

Discarded appliances (refrigerators, toilets, sinks) Tin cans, Metal containers

Plastic tarps

Holes in concrete, cinder blocks

Other (Describe: Depressions in ground/floor, boats, pots, pans, etc.) 1.

# of larvicide tablets (Bti) deployed: ______

Surveyor signatures: ______57

IDENTIFICATION COUNT DATA # CONTAINER TYPE DET. DATE GENUS SPECIES SUBSPECIES FEMAL MALE Entry E ♀ ♂ (Tracker)

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Annex 6.1. Catch-bag Removal (BG-Sentinels, CDC Light Traps, CDC Gravid Traps) and Transportation to Lab Protocol.

Revision 1

Purpose: to effectively collect adult mosquitoes caught by BGSts and to transport to laboratory for either (1) taxonomic identification and storage only, and/or (2) pathogen testing.

Equipment to bring to the field when collecting samples: Cooler with ice or dry ice; extra catch bags for replacement on the traps; new fully charged batteries for replacement; field data collecting form (Annex 5.1); maps with markings of where traps were installed; a cell phone with correct time or a watch

Step 0. Before going to the field, ensure a catch-cup is readily available. To make a catch-cup:

1. Grab a medium-sized basic kitchen food storage container (lunch size): height of should be about 1.5x the height of the cryovials. 2. Cut out a piece of cardboard such that it is the diameter of the top surface opening of the container. 3. Puncture/cut out holes such that cryovial body fits snuggly through the hole, but the cap porting does not fall through. 4. Have cryovials placed in these holes to prepare for transfer of specimens after surveying the traps. Proceed to Steps 1 through 2b. Step 1. Remove mosquitoes from the trap by carefully removing the catch bag (Annexes 4.1, 4.2, 4.3). Ensure the label is also transferred to catch bag to avoid loss of data recording information.

Determine what the trapping is for:

Step 2a. If the trapping is for taxonomic identification purposes only:

1. Store the catch bag in a standard freezer for at least an hour to knock out the mosquitoes. 2. Proceed to species identification (Annex 4.6). 3. For short-term storing, storage of specimens in temperatures just below freezing may suffice (Annex 4.6).

Step 2b. If the trapping is for pathogen testing (and including taxonomic identification):

1. Maintain the cold chain from the field to the lab and limit freeze-thaw cycles (each freeze-thaw cycle will kill about half the virus). Transport catch bag from the field in a cooler either with cold packs or on dry ice. Minimize the travel time on the road. If the field party is visiting multiple sites, bring the catch bags back to the lab before going to next sampling site. 2. Make sure the petri dishes contain the same labels as the catch bag to ensure accurate data recording. Do not overcrowd petri dishes to avoid damaging specimens (e.g. loss of scales). Sort and identify specimens on a chill plate or tray of ice. Ensure only female mosquitoes species are kept. Toss out all other insect species and males. Also count, record, and discard dried up female specimens, such that only female mosquitoes in adequate condition for identification are kept. Female mosquitoes are to be sorted and placed in pools for arboviral testing of no more than 50 specimens; pools should be of a minimum of 12 specimens; each pool is for ONE vector species only.

3. After sorting of specimens, grab the catch-cup. Transfer using a small plastic funnel each mosquito pool in an individual plastic (not glass) screwcap cryovial fitted with O-rings to prevent contact

with CO2 during transport and storage. NOTE: If by-catch is frozen to mosquitoes, do not try to disentangle. Do not overfill cryovial. 4. Again, ensure all label information remains in each pool and is not lost in the processing chain. This may be done by externally labeling each vial using a permanent, ethanol-safe marker before chilling of the vial. Optimal sampling effort are 10 pools of 50 females of each vector species from each site per week to detect minimum infection rates (MIRs) ranging from to 20 per 1,000 females tested. o If arbovirus screening is not done immediately after sorting and pooling, pooled samples should be stored frozen, optimally at -80 degrees C. NOTE: If live females are kept overnight (and longer) from time of collection from trap to time of processing, feed them 5-10% sucrose.

Annex 6.2. Transport of Ovitrap Paddles to the Lab and Hatching of Eggs Protocol.

Revision 1

Purpose: To outline how to pick up paddles from ovitraps in the field, transport ovitraps paddles to the lab, and hatch the eggs. The protocol of hatching of the eggs was produced and shared by the Yale Peabody Museum.

Equipment: Eyeglass; plastic food storage container with punctured holes on lid; mosquito rearing chamber; fish food; and scissors;

Step 0. Pick up the paddles from the field: given that DEH uses wooden tongue depressors as ovitraps paddles and the high humidity levels of Guam, it is best to change paddles during each visit, unless the field staff decides the paddle is not wilted and/or not harboring any mold. If the paddle is crowded with eggs, the paddle should also be changed.

1. Place the paddles with eggs in the food storage container; do not overcrowd. 2. Replace the removed paddle with a new one if necessary; ensure the rubber band is not dried Tupperware out.

Step 1. Processing the paddles in the lab: remove paddles from the food storage container. If the paper towel is very wet, allow it to dry 20 – 30 minutes in the open air or in a larger food storage container to ensure no other insects will eat the eggs.

Step 2. When the paper towel sufficiently dry, remove the rubber bands. Spread the paper towels to allow more drying.

Step 3. When sufficiently dry, count the eggs under the microscope, and record number of eggs for each paddle. If the egg appears to not be viable, count the egg anyways.

Step 4. It is best that eggs be hatched in the following hours and/or days. Proceed to the following steps for egg-hatching.

Step 5. Open the mosquito rearing chamber and fill the bottom portion with 400 to 500 mL of water (spring water, distilled water, or dechlorinated tap water). Add one small pinch of mosquito food to the water and stir to distribute the food. Allow the water to sit for at least an hour before adding the eggs.

Step 6. Cut a slice of the filter paper with mosquito eggs. To avoid overcrowding, separate them into new chambers or jars within a few days.

Step 7. Slip the paper into the water so that it sinks to the bottom of the rearing chamber. If the paper floats, tap it with forceps to dislodge the air bubbles.

Step 8. Replace the chamber top, making sure that the funnel portion points upward into the top section, not into the section with the water.

Step 9. After 24 to 48 hours, all viable eggs will have hatched. Use forceps to remove and discard the filter paper. Rinse off the filter paper to make sure no larvae are clinging to it. If the filter paper is not removed, it will disintegrate and cloud the water.

Step 10. Open the chamber and add a small pinch of food every day if the larvae have eaten most or all of the food from the previous day. If not, wait and feed the next day. Once there are many pupae, add one final pinch and then do not feed again. Pupae do not eat and will soon emerge as adults.

Step 11. It is better to underfeed than overfeed. The water should never look dirty, cloudy, or brown.

Step 12. For the purpose of mosquito identification, waiting for the emerged adults to die on their own is acceptable. Ideally, freeze living adults for 30 min to 60 min to neutralize for identification.

Step 13. If the adults are to be kept alive for a while, soak a cotton ball in 10% sucrose solution honey. Place the cotton ball on the mesh screen. Adults will drink this “nectar” and live for a few weeks. Replace or re-saturate the cotton ball every few days. The sucrose will also aid in attracting newly emerged adults to the top chamber.

Annex 7. Enhanced Mosquito Surveillance Activities for Levels 1, 2, and 3.

Risk Surveillance Activities* Objectives Level • Ensure usual Tier 1 sites surveillance activities • Confirm non-endemic acquisition of are maintained as outlined in Annex 3.1. arboviral/parasitic infection, • Set-up five ovitraps around case-patient’s • Assess container litter level around home (200-meter radius), patient home, • One BGSt near case-patient home, and 1 day • Assess effectiveness of vector control

Level Level 1 of larval/pupal survey around home before measures deployed, vector control activities, and 1 day after vector • Quantify abundance of Ae. albopictus control activities. collected, • Opportunistic larval/pupal survey. • Ensure Ae. aegypti not present. • Ensure usual Tier 1 sites surveillance activities • Quantify abundance of local vectors are maintained as outlined in Annex 3.1, and: of diagnosed illness (arbovirus or o Add two to three BGSts at AWPI parasitic): Airport and Piti Commercial Port o If arboviral: Ae. albopictus, • Set-up five ovitraps around case-patient’s and o If parasitic: Anopheles spp. neighbours’ homes (200-400-meter radius), (malaria), Culex • One BG-Sentinel near case-patient home, and quinquefasciatus (filariasis). 1 day of larval/pupal survey around home • Assess effectiveness of vector control

Level Level 2 before vector control activities, and 1 day after measures deployed. vector control activities, • Determine presence or absence of Ae. • One BG-Sentinel near neighbours’ home, aegypti. • Additional ovitraps (and/or BGSts if possible) • If resources permit: conduct mosquito around nearby high- and moderate-risk sites, pathogen testing on collected • Opportunistic larval/pupal surveys around samples, insecticidal resistance patient’s home. testing, • Map out case patients’ homes and assess • Conduct mosquito pathogen testing proximity to each other. on collected samples imperatively. • Ensure usual Tier 1 sites surveillance activities • Conduct insecticidal resistance are maintained as outlined in Annex 3.1, and: testing. o Add two to three BGSts at AWPI • Quantify abundance of local vectors Airport and Piti Commercial Port of diagnosed illness (arbovirus or • Place BG-Sentinel around each case-patient parasitic): home, o If arboviral: Ae. albopictus, • Conduct larval/pupal survey around each case- o If parasitic: Anopheles spp.

Level Level 3 patient home 1 day before vector control (malaria), Culex measures, and 1 day following vector control quinquefasciatus (filariasis). measures deployed, • Assess effectiveness of vector control • Add ovitraps and BG-Sentinels around nearby measures deployed, high- and moderate-risk sites (as many as • Determine presence or absence of Ae. feasible), focusing on nearby schools and aegypti: declare public health health facilities. emergency if Ae. aegypti is detected.

* If a malaria/filarial case is reported, CDC Light Traps should also be used to ensure capture of Anopheles/Culex.

Annex 8. Enhanced Surveillance Activities for Detection of Exotic Species Import on Guam.

Scenario Surveillance Activities Objectives • Communicate weekly with military base • Enable detection of Ae. aegypti having detected Ae. aegypti, outside military enclosures if it

• Ensure Tier 1 sites surveillance activities exists. conducted as outlined in Annex 3.1, • Evaluate extent of artificial water- o Add two to three BGSts at AWPI holding containers around military Airport and Piti Commercial Port. bases. Scenario1 • Conduct larval/pupal surveys in high- and • Ensure full-proof surveillance at moderate-risk sites near military base ports of entry. having detected Ae. aegypti. • Declare public health emergency for • Determine extent of Ae. aegypti Government of Guam to launch the EOC to occurrence on Guam. respond to threat with DoD, • Determine which site(s) and • Ensure usual Tier 1 sites surveillance region(s) of the island at highest activities are maintained as outlined in risk of arboviral transmission. Annex 3.1, and: • If at least 50 female Ae. aegypti

o Add two to three BGSts at AWPI specimens are collected, conduct Airport and Piti Commercial Port. pathogen testing on pool. o Conduct larval/pupal surveys at all Tier 1 sites. Scenario2 • Ensure all Tier 2 sites surveillance are conducted as outlined in Annex 3.2, • Conduct larval/pupal surveys around site(s) of detection of Ae. aegypti, • Deploy as many BGSts as feasible in and around sites with recorded presence of Ae. aegypti. • Communicate finding with military and CDC, • Determine extent of exotic species • Ensure usual Tier 1 sites surveillance occurrence on Guam.

activities are maintained as outlined in • Assess presence and extent of

3 Annex 3.1, and: favoured egg-laying water-holding o Add two to three BGSts at AWPI containers. Airport and Piti Commercial Port. • Conduct pathogen testing on exotic

Scenario o Conduct larval/pupal surveys at all species specimens if at least 50 Tier 1 sites. specimens are collected. • Conduct larval/pupal surveys around site(s) of detection of exotic vector.

A new mosquito species record in a given region such as the island of Guam may be considered ‘incidental’ (i.e. an ‘escape’) or established. The degree to which the species is established may vary. Generally, a species is considered “established” if multiple life stages are collected, the species is found across different sites/locations, and thus, larvae/pupae collected from more than one water-holding container.

Annex 9. Routine Source Reduction Activities and Community Mobilization.

Table 1. Source reduction activities conducted by DEH and by general population.

Site Types* DEH Source Reduction Activities Community Source Reduction Activities Residential • Eliminate artificial rubbish in/outside and • Put pressure on village mayor homes around homes, to rid them of discarded • If rubbish can’t be eliminated, flip over to kitchen appliances, prevent flooding, • Maintain property clean or • Remove tires or cover, fill in tires with cover with a shelter their gravel, or pierce holes in tires at 6, 9, 12, personal rubbish, and 3 o’clock) • Targeted removal of artificial • Opportunistic campaigning: speak to water-holding containers are residents about keeping their particularly favored by neighbourhood clean, especially during mosquitoes (tires, bromeliads, pre-high-risk season. drums) Large urban • Suppress vector breeding with monitored N/A. infrastructures larviciding if larval surveys indicate a (e.g. water productive mosquito breeding site. tanks, rainwater collection tanks, gutters, culverts) Construction • Inspect and determine risk level posed by • Construction workers: actively sites and site, limit standing water. dump yards • Educate construction workers about limiting standing water accumulation on construction site. Schools, • Eliminate artificial water-holding • School maintenance personnel health containers in and around schools and & school children: weekly facilities facilities, clean-ups inside/outside • Speak to school director or health facility facility. personnel about weekly clean-ups within the school or health facility. AWPI Airport, • Eliminate artificial water-holding • Airport personnel: eliminate Piti containers inside/outside ports of entry, artificial water-holding Commercial • Educate personnel about looking out for containers on a regular basis. Port containers and eliminating containers. Hotels • Engage hotel personnel to eliminate water- • Make rubbish elimination an holding containers from hotel property. integral part of hotel landscaping. o Engage hotel gardening/landscaping staff. *Large urban areas should be prioritized because the population density is greater than in rural areas, which inevitably fosters a more likely source of vector importation.

Table 2. Known productive natural and artificial water-holding containers.

Artificial Containers Natural Containers Tires Coconut husks, shells Buckets, Drums Tree holes Flower pots Crab burrows Ground pools, Tarpaulin depressions Rock pools Styrofoam containers Bromeliads

Recommendation: the engagement of personnel of schools, health facilities, and hotels in elimination of water-holding rubbish is most effective in concordance with law enforcement. Thus, it is highly advised that the DEH work to implement citation procedures should personnel not abide to DEH legislation.

Annex 10. Enhanced Vector Control for Disease Outbreaks and Mitigation of Human-Vector Contact.

Table 1. Enhanced Vector Control for Disease Outbreaks and Timing.

Risk Enhanced Vector Control Activity Timing of Activities Level/Scenario • Level 1 • Eliminate water-holding containers around 1 day following mosquito • Level 2 case-patient home, collecting around case-patient • If the patient lives near large water-holding home.* infrastructures (e.g. large refuse), use larvicides. Level 3, • Eliminate all water-holding containers around 1 day or immediately after Scenario 1 case-patients’ homes, neighbours, and nearby mosquito collecting conducted high- and moderate-risk sites, around case-patients’ homes. • Communicate with military base to ensure removal of water-holding containers within military base, • Apply IRS in case-patients’ homes, and in homes 200 m from case-patients’ homes, • Use larvicides in large water-holding containers around case-patients’ homes Level 3, • Declare public health emergency for • Declare public health Scenario 2 Government of Guam to launch the EOC to emergency as soon as respond to threat with DoD, detection is confirmed, • Eliminate all water-holding containers around • On same day, conduct case-patients’ homes, neighbours, and nearby extensive mosquito high- and moderate-risk sites, collecting around site of • Eliminate all water-holding containers around detection of Ae. aegypti, location of Ae. aegypti detection, • Follow collecting by • Communicate with military base to ensure enhanced vector control removal of water-holding containers within activities, military base, • Conduct collecting 1 day • Apply IRS in case-patients’ homes, and in following vector control, homes nearby location of Ae. aegypti • Source reduction and IRS detection (incl. and in homes 200 m from application should be case-patients’ homes), applied again if follow-up • Use larvicides in large water-holding mosquito collecting yield containers around case-patients’ homes and additional Ae. aegypti or if around location of Ae. aegypti detection mosquito populations not suppressed. Level 3, • Eliminate all water-holding containers around • Conduct extensive Scenario 3 case-patients’ homes, neighbours, and nearby mosquito collecting around high- and moderate-risk sites, site of detection of exotic • Eliminate all water-holding containers around mosquito species, location of initial exotic mosquito species • Follow collecting by detection, enhanced vector control activities,

• Communicate with military base to ensure • Conduct collecting 1 day removal of water-holding containers within following vector control, military base, • Source reduction and IRS • Apply IRS in case-patients’ homes, and in application should be homes nearby location of exotic mosquito applied again if follow-up species detection (incl. and in homes 200 m mosquito collecting yield from case-patients’ homes), additional exotic mosquito • Use larvicides in large water-holding populations not containers around case-patients’ homes and suppressed. around location of exotic mosquito species detection. *It is essential that mosquito collecting be conducted prior (day before, or hours before) to launching of enhanced vector control activities. This is to increase the DEH’s likelihood of detecting Ae. aegypti if present, and/or inferring the likely vector of the outbreak (if applicable).

Table 2. Mitigation of Human-Vector Contact.

Activity Target Population Mosquito topical repellents Underprivileged communities. distribution (DEET). Mosquito nets. Pregnant women (for zika). Mosquito proofing of windows Schools, health facilities. and doors.

Annex 11. CalSurv Terms and Conditions.

Provided by UC Davis DART group Principal Investigator.

Summary of Gateway Modules

1. Customizing the Gateway for Your Agency • User Settings This section allows users to modify preferences for their personal profile, such as user name and password, contact email and communications preferences, date and time formats, as well as many other options. • Agency Settings Users with administrative privileges within their agency may also update agency settings, including the agency name, agency code, address, and other contact information. Users with administrative privileges may also modify roles (permissions) of other users within their agency, and add or deactivate user accounts.

2. Data Entry and Management • Surveillance Sites The “Sites” module allows users to define and manage established locations for routine surveillance. Many details can be recorded, but minimally, site information consists of a site code and defined geographic location for the site. The location can be defined using either a known latitude and longitude or by selecting a point location on the Google Map. Site names are optional, but we recommend assigning a unique name to each site that is familiar to other users within the agency. It is also useful to include the site address when available to validate the geographic coordinates in case of discrepancies. • Arthropod Collections and Pools The “Arthropod” module is for recording and managing data on arthropod collections or pools of arthropods to be tested for viruses or other parasites. Collections typically involve either trapping arthropods using one of many trap types or site inspections by agency personnel.

• Sentinel Animals The “Sentinel” module allows for recording the collection and testing of blood samples from sentinel chickens. These samples are tested for serological

evidence of arbovirus exposure, and test results can be tracked using this module. • Carcasses The “Carcass” module is used for documenting dead birds reported by the public. Due to the high mortality that West Nile virus causes in some bird species, dead birds often provide evidence of West Nile virus activity in an area. This module also allows agencies to record test results for dead birds that are tested for WNV by RTPCR. • Insecticide Applications The “Application” module allows agencies to set up the equipment, technicians, and pesticide mixes that are applied within an agency, using EPA-registered products. Mosquito sources are defined by drawing points, lines, or polygons on the map, and applications involve application of pesticide to the defined sources at a particular time. All spatio-temporal information is recorded in the Gateway database, and at the agency’s option, this module offers automated reporting of pesticide use to county agricultural commissioners to comply with state regulatory requirements. • Insecticide Resistance The “Resistance” module tracks tests for insecticide resistance, including the resistance test results, source location of the mosquitoes tested, and the date of collection. At this time, the types of resistance testing that can be recorded in the Gateway are larval bioassays, CDC bottle bioassays, and PCR for KDR mutations.

3. Data Export A basic premise of the CalSurv Gateway is that all data entered into the system can be exported as spreadsheet-style output for use by each agency. Data are exported by data type, and any registered Gateway user can export data for his or her agency. Filters can be applied to limit the download to only the data desired by the user.

4. Tools and Maps • Arthropod Abundance Anomaly: This tool produces a plot showing a particular year’s abundance pattern compared to that of the 5 preceding years. Abundance is expressed as mosquitoes per trap-night (or day), and the data can be grouped by time period (week, half-month, month, or custom interval), spatial location, trap type, species, or other attributes. The data associated with the graph can be downloaded in spreadsheet format for further analysis. • Arthropod Pool Infection Rate: This tool produces a plot showing trends in estimated prevalence of a pathogen (e.g., WNV), along with a statistical confidence interval. Data can be grouped by time period (week, half-month, month, or custom interval), spatial location, trap type, species, or other

attributes. The data associated with the graph can be downloaded in spreadsheet format for further analysis.

• Risk Assessment: This tool implements the risk assessment model from the California Mosquito-Borne Virus Surveillance and Response Plan. The risk assessment includes a variety of surveillance indicators, and the Gateway’s calculator integrates all available surveillance data to provide an estimate of overall risk for human infection with West Nile virus. Data can be grouped by time period (week, half-month, month, or custom interval), spatial location, trap type, species, or other attributes. • Vector Index Calculator: This tool calculates the product of female mosquito abundance and infection prevalence to provide an estimate of the abundance of infected female mosquitoes. The data can be grouped by spatial location, trap type, species, or other attributes. The data associated with the graph can be downloaded in spreadsheet format for further analysis.

5. Forwarding data to CDC’s ArboNet and MosquitoNet The CalSurv Gateway has established mechanisms for semi-automated reporting to arbovirus surveillance data to CDC’s ArboNet database and for reporting of collections of Aedes aegypti and Aedes albopictus to CDC’s MosquitoNet database. For both databases, a designated person, either for a single agency or a broader region such as a state, logs into the Gateway and submits the data after reviewing it for accuracy. This is typically done monthly, and the user can define filters (e.g., by date) for the data to be submitted. This feature is only available to personnel authorized to submit data on behalf of the agency or state, and the submitting user must also receive authorization to submit data from CDC.

Contacts: Christopher M. Barker, Ph.D. (Principal Investigator) +1 530 752 0151 [email protected] Jody Simpson (Data Steward) +1 530 752 8380 [email protected]

California Vectorborne Disease Surveillance Data Policy

March 01, 2013

Introduction

What follows is a policy statement that outlines permission to use electronically-stored vectorborne disease surveillance data for multiple uses and by various organizations. The proceeding guidelines are intended for agencies that participate in the California Vectorborne Disease Surveillance Program (CalSurv), a joint activity of the Mosquito and Vector Control Association of California (MVCAC), the California Department of Public Health (CDPH), and the University of California (UC). This policy is administered by the CalSurv Steering Committee, comprised of members from the three agencies listed above.

Preamble

Over the past decade, the migration from locally-maintained paper surveillance data records to centrally-stored electronic records has raised questions concerning the ownership, use, release, and publication of data. Ownership issues were clearer in the past because data were generated by relatively few individuals and organizations and they were retained in formats that were not easily shareable. However, as the goal of centrally-coordinated collection and testing of surveillance specimens has come closer to realization, it has also become apparent that a set of guidelines is required to address questions of stewardship of the data, availability of the data for analysis and publication, acknowledgement of data origination, and permission for use of the data.

Guiding principles

1. Data generated as a result of publicly supported programs are public property and will ultimately become part of the public record. However, unrestricted, immediate availability of all data is not guaranteed and should not be expected.

2. Permission is required before surveillance data may be used for any purpose. Uses for which permission is required include, but are not limited to: publication or presentation in any written, oral, or electronic format; collation, summarization, or analysis; redistribution to secondary parties.

3. In the interest of fairness and protection of individual privacy, availability of these data is subject to restrictions, including but not limited to the release of names, addresses, phone numbers or other contact information, and other personal identifiers associated with disease cases and related surveillance. Where there are privacy concerns,

certain private information may be redacted, when present, from the original records before they are provided to a requestor.

4. Persons intending to aggregate, analyze, or publish these data are required to credit in the form of a citation acknowledging those individuals and organizations most responsible

for the creation and collection of said data and, in the case of ongoing data programs, those individuals and organizations most responsible for organizing and maintaining the mechanisms by which data are assembled.

Example: Data obtained through CalSurv Data Request #00001 on 1 January 2008: California Vectorborne Diseases Surveillance System.

5. Because of the impracticality of publicly acknowledging individual agencies and their employees in wide-ranging studies, a blanket permission request may be made in these cases and submitted to CalSurv. The guiding principles listed here must be acknowledged and accepted by the inquiring party.

6. Any individual (“requestor”), regardless of parent agency affiliation, must obtain permission to use surveillance data prior to any intention for presentation, publication or redistribution. However, exceptions exist for the following situations (all exceptions terminate upon the individual’s separation from their parent agency):

a. An individual need not request permission to use data generated solely by his or her parent agency. (Permissions for access still may be needed within the individual’s agency, but are beyond the purview of CalSurv and not addressed in this document.) b. An individual appointed by CalSurv as the Data Steward. This appointment is automatically granted to the server administrator overseeing all pertinent datasets and is limited in scope to only the distribution of requested datasets approved by the CalSurv Steering Committee.

7. Requestors must be directed to the CalSurv Data Steward for procedures to formally request use of such data.

8. Requests for use of CalSurv data will be considered by the CalSurv Steering Committee. Individual agencies whose data may be part of the request may provide opinions to be considered by the CalSurv Steering Committee. A request requires two- thirds of the committee to vote for approval. Written request approvals will be forwarded to the Data Steward for fulfillment. Once the Data Steward compiles the requested dataset, the steering committee will inspect the data before delivery to the requestor.

9. The intellectual property rights to the surveillance data belong to the originating agencies participating in the California Vectorborne Disease Surveillance System. Any decisions made by the CalSurv Steering Committee do not transfer, release or grant any intellectual property rights to the requestor. The only permitted exception is if the transfer, release or granting of rights is explicitly written in the request approval.

Limits of the Data Policy

1. This policy applies only to those datasets managed by CalSurv. These datasets are: a. Arboviral surveillance and infections i. sentinel animals (e.g. chickens) ii. dead birds and squirrels iii. mosquitoes. 2. Datasets exempt from this policy are: a. Summaries of surveillance measures needed by agencies such as CDPH to satisfy statutory requirements for agency reports. Permission to use data from surveillance datasets not stated in this policy must be sought from the agencies possessing the data. Also, surveillance datasets from agencies located outside California are not within the purview of the CalSurv Steering Committee and permission to use such data must be sought from the agencies possessing the data.

Annex 12. Monitoring and Evaluation for Mosquito Surveillance and Control.

Table 1. M&E framework for mosquito surveillance.

Activity Focal Unit Timing/Frequency of Scope of Activity Indicator Data Type for Activity Indicator Sentinel sites (Tier 1) DEH MSCP. As outlined in Annex • Track mosquito 1. No. of Sentinel 1. Quantitative. surveillance. 3.1. population sites, 2. Quantitative: No abundance 2. Weekly of Sentinel site trends, frequency of visits per week. • Confirm Sentinel sites presence/absenc surveillance. e of Ae. aegypti in high-risk areas. Tier 2 and Tier 3 DEH MSCP. As outlined in • Track mosquito 1. No. of Tier 2 and 1. Quantitative. surveillance. Annexes 3.2, and 3.3. population Tier 3 sites, 2. Quantitative: No abundance 2. Bi-weekly (Tier 2) of Tier 2 site trends, count of Tier 2 visits per 2 • Confirm surveillance, weeks. presence/absenc 3. Bi-annual (Tier 3) 3. Quantitative: No. e of Ae. aegypti count of Tier 3 of Tier 3 site in moderate- and surveillance. visits per year. low-risk sites, • Confirm site status as Tier 2, and Tier 3 sites, respectively. Adult mosquito DEH MSCP. As outlined in To determine: 1. No. of adult traps 1. Quantitative. surveillance. Annexes 3.1, 3.2, 3.3. • Transmission deployed per 2. Quantitative. risk-levels, week, • Presence/absenc 2. No. of sites e of important which have adult vector species, traps each week.

• Effectiveness of vector control strategies. Larval/Pupal DEH MSCP. As outlined in To determine: 1. No. of 1. Quantitative. surveillance. Annexes 3.1, 3.2, 3.3. • Productive egg- larval/pupal 2. Qualitative: laying sites, surveys Yes/No. • Effectiveness of conducted each vector control week, strategies 2. Calculation of entomological indices. Egg surveillance. DEH MSCP. As outlined in To determine vector No. of ovitraps Quantitative: Annexes 3.1, 3.2, 3.3. presence/absence. deployed weekly. Ovitraps/week.

Mosquito species DEH MSCP Lab Weekly. • To determine 1. No. of 1. Quantitative: identification. Responsible/Entomol species mosquitoes Mosquitoes ogist. composition, identified per identified/week, abundance, and week. 2. Quantitative: trends. 2. No. of shipments Shipments to • To confirm to WRBU per WRBU/month. presence/absenc month. e of Ae. aegypti. • To track abundance and trends of Ae. albopictus. Mosquito pathogen- DEH MSCP Lab As needed. To determine No. of mosquito Quantitative. testing. Responsible/Entomol presence/absence of pools tested ogist. viral RNA in local annually. mosquito population. Mosquito DEH MSCP Lab Monthly. To evaluate local No. of CDC bottle Quantitative. insecticidal Responsible/Entomol insecticidal bioassays conducted resistance testing. ogist. resistance status. monthly.

Table 2. M&E framework for mosquito control.

Activity Focal Unit Timing/Frequency of Scope of Activity Indicator Data Type for Activity Indicator Elimination of egg- DEH MSCP Field As outlined in Reduce presence of No. of larval/pupal Quantitative laying sites. Team. Annexes 7, 8, 9. artificial and natural surveys conducted water-holding per month. containers. Distribution of DEH MSCP Field As outlined in Reduce larval/pupal 1. No of larvicide 1. Quantitative. larvicides. Team. Annexes 7, 8, 9. populations in tablets used 2. Quantitative. containers that monthly. cannot be removed. 2. No of house inspections annually. IRS. DEH MSCP Field As outlined in For immediate No. of IRS activities Quantitative. Team. Annexes 7 and 8. response to reported conducted annually. arboviral cases, and/or detected presence of Ae. aegypti. Public outreach. DEH MSCP. As outlined in To promote public 1. No. of pamphlets 1. Quantitative. Annexes 7, 8, 9. awareness and distributed per 2. Quantitative. mobilization for month, 3. Quantitative. personal protection 2. No of household 4. Quantitative. against mosquitoes inspections and for elimination annually, of egg-laying sites at 3. No. of “mosquito home. days/months” annually. 4. No. of DEET cans distributed.